Effects of polyisoprenoids from Avicennia lanata and Avicennia alba leaves on the gene expression of PI3K, Akt1, mTOR, P53, and EGFR in human colorectal adenocarcinoma WiDr cells using reverse transcription-PCR [version 1; peer review: 1 approved with reservations]

Background: Mangrove plants produce a polyisoprenoid compound. Polyisoprenoids have been proven to have anticancer properties. This study investigated the inhibitory activity of polyisoprenoids derived from the leaves of mangrove plants Avicennia alba and Avicennia lanata regarding the expression of PI3K, Akt1, mTOR, P53, and EGFR genes against human colorectal adenocarcinoma WiDr cells. Methods: Anticancer activity


Introduction
Cancer is a disease characterised by uncontrolled cell growth.Cancer cells can evade apoptosis and avoid signals that suppress its growth, impede the ability to form new blood vessels (angiogenesis), and halt its invasion and metastasis 1 .According to the Global Cancer Observatory, in 2018, Asia had the highest incidence of colon cancer with 51.8% of the global cases.Colon cancer is one of the top three causes of death in the world 1 .The use of chemotherapeutic agents constitutes a treatment for colon cancer, in addition to surgery and radiation therapy.Chemotherapeutic agents generally suppress the growth or proliferation of cancer cells, simultaneously causing toxicity in the body 2 .
Natural ingredients developed as potential chemotherapeutic agents include mangrove leaves.Mangroves are vegetation formations found in littoral areas in tropics and subtropics 3 .Polyisoprenoids are secondary metabolites found in several mangroves, distributed as dolichol and polyprenol on the leaves and roots of mangrove plants 4 .So far, few studies have reported the pharmacological activity of polyisoprenoids obtained from mangrove species.Thus, it is essential to study the potential and mechanisms of polyisoprenoids in mangroves as a natural ingredient for anticancer pharmaceuticals and medication 4 .For instance, methanol extracts of Avicennia alba (bark and leaves) present anti-proliferative activity in MCF-7 and T47D 5 .Additionally, this extract has cytotoxic effects on a variety of cancer cells, including colon cancer cells -HT-29 6 .Previous research suggests that polyisoprenoids induce cancer cell cycle inhibition in adenocarcinoma of the colon (COLO 320 HSR, WiDr, and LS174 cells) in the G2/M phase and reduce the percentage of Bcl-2 and Bcl-xL 7 .Polyisoprenoids have been previously reported as chemopreventive agents for colon cancer 8 , given that polyisoprenoids in A. lanata leaves have displayed anticancer activity for the same 9 .On the other hand, A. alba contains polyisoprenoids that induce cell cycle, apoptosis, and gene expression of COX-2 in colon cancer cells WiDr 10 .This extract has a mechanism for inhibiting the cell cycle at the G0-G1 phase, and apoptotic analysis occurs in the early phase of apoptosis in WiDr cells 9,10 .
The present study analysed the effect of immune-related genes' expression on WiDr cells in vitro using reverse transcriptionpolymerase chain reaction (RT-PCR).RT-PCR was developed as an in vitro test to measure the biological activity of plasmid DNA-based products (pDNA).The said test measures RNA-specific transgenic messengers (mRNA) derived from transfected cultured cells.Forward and reverse primers have been designed to trigger selective RT-PCR reactions for plasmid mRNAs and differentiate between the level of individual plasmid expression in multivalent pDNA 11 .Therefore, the present study aims to investigate the inhibitory activity of polyisoprenoids obtained from the leaves of mangrove plants A. alba and A. lanata concerning the expression of PI3K, Akt1, mTOR, P53, and EGFR genes in human colorectal adenocarcinoma WiDr cells.

Plant material
The leaves of two mangrove species -A.alba and A. lanata were collected from the village of Lubuk Kertang, Brandan Barat, Langkat, North Sumatra, Indonesia.The sample site is situated at 04° 07' 39.71'' North latitude and at 98° 30'97.87''East longitude.

Preparation of isolation polyisoprenoid alcohols
Every 500 g of powdered simplicia mangrove leaves of A. alba and A. lanata was macerated with a mixture of chloroform/methanol (2:1, v/v) (CM21) for 48 h.The cell wall debris insouble in CM21 was removed by filtration paper (Advantec, Japan) and the extract was partially purified as lipid extract.The simplicia procedure is described briefly; the wet mangrove leaves were sorted to remove dirt; the leaf bones were removed and cleaned from attached leaf spines; the leaves were cut into 3 cm long pieces, after which they were washed under running water, drained, and weighed.Subsequently, the mangrove leaves were dried in a drying cabinet, dry sorted to separate unwanted plant parts and other impurities that were still present, remain in dry simplicia, and weighed and stored in tightly closed plastic containers 8 .The lipid extracts of leaves were refluxed at a temperature of 65°C for 24 h in 86% ethanol containing KOH 2 M. The portions were further saponified and diluted with 2 mg/mL n-hexane.The extract in n-hexane was concentrated using rotary evaporator at 40°C.Subsequently, a thick extract was obtained and the concentration was adjusted to 1 mg/mL n-hexane.

Cytotoxicity analysis
Cytotoxicity tests were conducted on the WiDr and Vero cells using the MTT method.Cells were grown in 96 well microplates to obtain a density of 1 × 10 4 cells/well and incubated in a 5% CO 2 incubator at a temperature of 37°C for 48 h to ensure good growth.Once the new medium had been replaced, the cells were treated with varying concentrations of PAA and PAL (1000, 500, 250, 125, and 62.5 µg/mL) as previously reported 10 .5-Fu (Sigma Aldrich) was used as a positive control with the same concentration of PAA and PAL and incubated in 5% CO 2 at 37°C for 48 h.At the end of the incubation, the culture media was removed, and the cells were washed with PBS.In each of the wells, 100 mL of culture medium (RPMI) and 10 mL MTT (Sigma Aldrich) were added.The cells were incubated again for 3-6 h in 5% CO 2 at 37°C.The reaction was stopped with 10% SDS reagent (Sigma Aldrich) in 0.01 N HCl (Merck).The plate was wrapped to protect it from the light so that the wells were opaque, and it was left overnight at room temperature 14 .Absorption was measured by the ELISA reader (Benchmark 10431, BioRad) at a wavelength of 595 nm.
Selectivity index (SI) was determined from the IC 50 of the polyisoprenoid extract from PAA and PAL leaves in Vero cells versus WiDr cells to exhibit the cytotoxic selectivity of the polyisoprenoid extract, as previously reported 9 .IC 50 was calculated from concentrations that caused death among 50% of the cell population analysed using probit analysis in SPSS version 23 with a significance of 0.05 8 .

Cell cycle analysis
The WiDr cells (5 × 10 3 cells/well) were added to a 6-well plate which was incubated for 24 h for optimal growth.Subsequently, the cells were exposed to selected concentrations of PAL and PAA (1/5 IC 50 ) and incubated again 10 .Floating as well as attached cells were collected by adding 0.025% trypsin.The cells from each well were transferred to a separate eppendorf tube. 1 mL PBS was added and the PBS was removed with a micropipette and centrifuged at 2500 rpm for 5 min.The supernatant was removed and 1 µL RNase/PI staining solution (Thermo Fisher Scientific) was added and kept for 10 min a dark place (avoiding light) at 37°C.The cell cycle distribution was analysed using the FAC Scan Flow Cytometer (BD Biosciences) and the percentage of cells obtained in each cell cycle phase (G1/S and G2/M) was calculated using the software ModFit LT. 3.0 s for Windows (Verity Software House).

Apoptosis analysis
The double staining method was used to determine the level of apoptosis.WiDr cells were grown in a 6-well microplate at density of 5 × 10 5 cells/well and incubated for 24 h.The following day, the cells were treated with concentrations of PAA and PAL (1/5 IC 50 ) and incubated for 24 h.The procedure for apoptosis with double staining was conducted by taking cells from the CO 2 incubator and observing the conditions.Then, the calculated cells were used to prepare 24-well plates and slipcovers.200 µl of cell suspension was evenly and slowly transferred just above the coverslip.The cell was kept in the incubator for 3-30 min to attach to the coverslip with 800 µl of culture media which was added for 48 h of incubation.The culture media was slowly disposed, and the cells were washed with PBS (500 µl).The sample and media were added into the well for control cells and then incubated.All media from the well was slowly removed using a Pasteur pipette.Cells in the wells were washed with PBS.The coverslip was removed using tweezers, placed on a glass slide, and labelled.10 µl of the reagent mixture of ethidium bromide acridine orange (Sigma Aldrich) was added over the slipcover.The mixture was flattened and gently rocked.Apoptosis was observed under a fluorescence microscope (Olympus CKX41) 9 .The fluorescent green cells were alive and the fluorescent red cells were dead 15 .

Gene expression analysis
Total RNA was extracted from the WiDr cells treated with PAA and PAL (7.5 × 10 8 cells/well) using the Total RNA Mini Kit (Geneaid), according to the manufacturer's protocol.The total RNA (3 mg each) was reverse-transcribed with 1 µg random primer to produce cDNA in a total volume of 20 µl using ReverTra Ace kit (Toyobo) with 10 mM dNTP to incubate for 10 min at 30°C, for 60 min at 42°C, and for 5 min at 99°C according to manufacturer's procedure.The resulting cDNA mixture was diluted using 100 µL TE buffer (10 mM Tris/HCl, 1 mM EDTA, ph 8.0) and directly used for the subsequent PCR.

Statistical analysis
All the data were analysed using SPSS version 23.The data are presented as mean ± standard error of the mean (SEM).One-way variance analysis (ANOVA) was used to compare the results for different conditions.P < 0.05 was considered significantly different.

Cell cytotoxicity of PAA and PAL
The cytotoxicity test is a preliminary parameter to determine the potential toxicity of a test substance, particularly cancer cells.The toxicity is expressed by IC 50 parameters.However, the cytotoxic test can also be performed to assess the toxicity of a test substance on normal cells.So, it can be used to demonstrate selective cytotoxic effects against a cancer line.In this study, the test material is cytotoxic for the polyisoprenoids of leaves derived from two mangrove species (PAA and PAL) against colon cancer cells (WiDr) with a concentration series of 1000, 500, 250, 125, and 62.5 µg/mL.The purpose of testing the extract was obtaining the smallest IC 50 value for subsequent use as an advanced test for anticancer activity.The results showed that the smallest IC 50 value obtained from the polyisoprenoids of leaves from PAL was 243.32 ug/mL and from PAA was 258.14 ug/mL.Therefore, these concentrations were used in the remaining experiments of the study.The cytotoxic effects were indicated by absorbance values and analysed using a probit analysis to obtain IC 50 , as shown in Table 2.
The greatest cytotoxic activity against WiDr cells, shown by the smallest IC 50 value, was obtained from PAL.Therefore, PAL has the most active anticancer activity because the IC 50 value showed that PAL could block 50% of the WiDr cell growth.Some extracts are only considered active if they have IC 50 values ≤100 µg/mL 16 .However, it has been demonstrated that an extract value of IC 50 of 100-500 µg/mL can be classified as moderate and, therefore, can potentially be developed as an anticancer agent 17 .Even though a study has reported that an extract is considered active if IC 50 > 500 µg/mL 18 .As shown in Table 2, the SI of PAL and PAA were lower than that of the positive control.The cytotoxicity of PAL and PAA in the present study included an interesting SI against WiDr cells in a dose-dependent manner 17 .

Cell cycle analysis with PAA and PAL
A previous study showed that methanol and water extracts of A. alba leaves have distinctive properties in regulators and mediators of cancer 19 .This study tested the cell cycle using flow cytometry to determine the distribution of cells in each phase of the cell cycle at sub G1, S, and G2/M after treatment and obtained predictable pathway inhibition using PAA and PAL to inhibit the cycle cell 20 .
The inhibition of the cell cycle in this study is shown in Figure 1 and Table 3. Table 3 shows the control group's WiDr cell accumulation in the G0/G1, S, and G2/M phase as 76.63%, 7.22%, and 17.93%, respectively.The accumulation of cells in the S phase and G2-M cells increased by 10.60%, 10.51% and 23.84%, and 22.05%, respectively after being administered with a concentration of PAL 1/5 IC 50 and PAA with the concentration of 1/5 IC 50 .The phase change is considered related to the concentration.However, the overall mechanism of inhibition of the cell cycle for PAA and PAL occurred at S and G2-M phases.
As Table 3 illustrates, the administration of 5-Fu with 1/5 concentration IC 50 decreases the accumulation of WiDr cells in the G2-M phase at 6.42%.The increase in cell accumulation occurred in the G0-G1 phase -S was 88.12 and 9.52%.However, it can be confirmed that the overall mechanisms of cell cycle inhibition of 5-Fu (in the G0-G1 and S phases) had a different mechanism compared with PAL and PAA.Treatment of cancer cells with 5-Fu can accumulate cells at the G1 phase and at the beginning of the synthesis phase (G1/S arrest) 21 .However, the cell cycle's inhibitory activity using 5-Fu depends on the type of cancer cell.In colon cancer cells HCT-15 and HT-29, 5-Fu inhibited at the G2/M phase.5-Fu increases the expression of cyclin A, cyclin B, and CDC2, which is a regulatory protein in the G2/M phase 22 .The mechanism that mediates the activity in this phase needs to be explored further.In Lovo and WiDr cells, 5-Fu inhibits the cell cycle in the S phase 18 .This suggests that the activity of 5-Fu is not always associated with thymidylate synthase inhibitory activity, and the activity of 5-Fu in the cell cycle if used in a different cell needs to be researched further.The antineoplastic activity of PAL and PAA occur in the S phase of the cell cycle, which involves the possibility of bonding with the DNA through intercalation between the base pairs as well as the inhibition of the DNA and RNA synthesis 18 .

Apoptosis analysis with PAA and PAL
In the present study, increased apoptosis (seen using the reagent acridine orange-ethidium bromide through the fluorescence microscope) was obtained by a percentage increase in each of the phases.Apoptosis is generally characterised by different morphological characteristics and biochemical mechanisms that depend on energy 23 .Apoptosis usually occurs during development and aging and as a homeostatic mechanism to maintain the population of cells in the network.It also is a defence mechanism when cells are damaged by disease or harmful agents or during immune reaction 24 .Cytotoxic data test samples indicate the presence of a cytotoxic effect as shown in Figure 2. The green cells are the live ones while the red ones are dead.The range of red fluorescent cells represents the necrotic cells.
The present analysis used raster imaging to count the dead cells and control cells after observing them under a fluorescence microscope.In total, 95.15% green colour (living cells) and 4.84% red colour (dead cells) were observed in control cells.
The results of the control cells were observed to be green/alive cells.The green colour comes from the orange acridine penetrating the entire living cell with intact membranes and nuclei.In cells treated with PAL and PAA, there was a predominantly red colour which illustrates that the WiDr cells were dead.The orange colour is produced by ethidium bromide interacting with damaged cell membranes and nuclei 25 .The test results showed that the extract can inhibit the growth of cancer cells, especially in WiDr cancer cells.The inhibition capability through the mechanism of apoptosis can also be evidenced through testing and analysis of double staining flow cytometry.The results of both analyses can illustrate the mechanism of cell death caused by apoptosis both quantitatively and qualitatively.Thus, the induction of apoptosis shows that these treatments are a promising treatment for cancer.The Expression of PI3K, Akt1, mTOR, P53, and EGFR genes The measurement of the expression of PI3K, Akt1, mTOR, P53, and EGFR genes using RT-PCR produces the band illustrated in Figure 3. Gene expression density was quantified using a computerised system (Table 4).Table 4 shows significant differences between the treatment groups.Gene expression results on PI3K, Akt1, mTOR, P53, and EGFR differed significantly (p < 0.05).
In this study, RT-PCR showed that the anti-apoptotic gene expression of P53 increases compared to control, whereas the expression of pro-apoptotic genes (PI3K, Akt1, mTOR, and EGFR) tend to decrease.P53 is a tumour-suppressing protein that can affect the permeability of the mitochondrial membrane Besides from being a tumour suppressor protein, P53 also acts as a transcription factor for the activation of the expression of multiple target genes involved in various biological functions, such as apoptosis and cell cycle arrest 30 .The results of the present study show that PAL and PAA significantly increase the gene expression of P53 at high concentrations, and the increase in P53 gene expression is concentration dependant; the higher the concentration, the higher the level of gene expression.These results are consistent with previous studies stating that the expression of P53 protein increases apoptosis 31 .
The density of PI3K, Akt1, EGFR and mTOR gene expression was been significantly downregulated in treatment cells compared to the control cell in the present study.PAL administration demonstrated a more significant reduction in PI3K, Akt1 and EGFR gene expression than PAA and 5-Fu, while mTOR was downregulated more with 5-Fu than PAL and PAA.
The density of the P53 gene expression was significantly upregulated in the treatment cells compared to the control cell, and this was more significant with PAL than PAA and 5-Fu.

Conclusion
Overall, the present study confirmed that PAL and PAA can affect anti-apoptotic P53 gene expression by upregulating this gene than the controls, while the expression of pro-apoptotic genes PI3K, Akt1, mTOR, and EGFR were downregulated compared to the controls.In addition, PAL and PAA inhibited the WiDr cell cycle in later apoptosis (S and G2-M1).Therefore, this study confirms that the polyisoprenoids derived from A. alba and A. lanata leaves are promising chemopreventive agents for colon cancer.
This project contains the following data: -Underlying data for Table 2-Table 4.
-Uncropped, unedited images for Figure 2   b.Sig (P) < 0.05: There is a significant difference with the group PAL.
c. Sig (P) < 0.05: There is a significant difference with the group PAA.
d. Sig (P) < 0.05: There was no significant difference in the positive control group (5-Fu).and directly induce apoptosis without inducing the transcription of the target gene associated with apoptosis in advance 28 .This condition causes apoptosis when the gene Bax mRNA expression does not increase and, thus, the pathway of apoptosis by P53 has two paths to the mitochondria -directly and indirectly through the activation of the transcription of genes under it.P53 molecule is a tumour-suppressing protein found at a low level under normal conditions and has a short life span.P53 is activated when the cells are exposed to stimuli such as This manuscript describes elucidation of the biological activity of two polyisoprenoid-containing extracts isolated from leaves of two mangrove plant species Avicennia lanata and Avicennia alba.To meet the generally acceptable standard of the manuscript quality this preliminary study requires extremely extensive revision.Firstly, all the biological data were obtained using crude and poorly characterized extracts and consequently all these experiments should be performed once again with highly purified polyisoprenoid mixture (polyisoprenoid content at least 90%).Such purification procedure is doable within a reasonable time period and is not very difficult.Secondly, several methodological issues require clarification, e.g.selection of the drug concentration points, appropriate control experiments (composition of the cell growth media), number of replicates, methods of quantification of PCR results and statistical analysis of the data.Thirdly, interpretation of the obtained results seems not really clear, in particular results of the analysis of the toxicity should be reconsidered (see below).Finally, the Authors should carefully discuss the available literature data to document the novelty of their study.
A detailed list of comments follows.
This manuscript describes study on the biological effects of the polyisoprenoid-containing extracts isolated from leaves Avicennia lanata and Avicennia alba.All tests were performed for two cell lines, WiDr and Vero.Shown are data on the effect of PAA and PAL on the cytotoxic activity, cell-cycle progression and expression of the selected genes: PI3K, Akt1, mTOR, and EGFR.

General comments:
All the biological tests described in this study were performed using the crude mixture of lipids obtained after alkaline hydrolysis of the leaf extracts.The Authors did not provide the approximate polyisoprenoid content in the mixtures used.Such complex mixture of natural compounds could be used just for preliminary tests of their potential biological activity.What is more, data presented in this study in fact confirm the weakness of the strategy used -e.g.different effects of pro-apoptocic activity of PAA and PAL on the level of expression of analyzed genes might suggest that this is NOT the effect of polyisoprenoids (very similar profile for both extracts) but rather some other components of these mixtures.
In order to present any reasonable conclusion on the biological effect of polyisoprenoids highly purified polyisoprenoid samples (purity higher than 90% based on the HPLC/UV assay) should be used.For this reason the experiments have to be reproduced with purified polyisoprenoid samples.
In this context the title of the manuscript does not describe the real content of the work.
Data presented in Table 2 suggest that analyzed extracts exert higher toxicity against Vero than WiDr cells.Consequently the Selectivity Index is below 1.It raises the question whether analyzed extracts could be considered as candidates for anticancer drugs since they posses higher cytotoxic activity against normal than cancer cells.Authors should carefully comment on this observation.Keeping in mind that analyzed extracts were not pure these data do not preclude the potential usefulness of polyisoprenoids as drugs although this conclusion cannot be made on the basis of results shown in Table 2. Furthermore, interpretation of the quantitative results of cytoxicity against WiDr cells is questionable -the difference between both IC50 values obtained for PAA and PAL is approx.6% (Table 2); thus the conclusion presented by Authors seems highly exaggerated.Finally, why are no SD values are presented here?Have these experiments been performed only once?
Experiments on the effect of plant extracts on the cell-cycle progression are summarized as follows: 'The antineoplastic activity of PAL and PAA occur in the S phase of the cell cycle, which involves the possibility of bonding with the DNA through intercalation between the base pairs as well as the inhibition of the DNA and RNA synthesis.'Such conclusion definitely requires additional data supporting the suggested mechanism(s).Moreover, presented data were not analyzed using statistical method.Fig. 2 presents representative data but how many biological replica were performed?How many microscopic snapshots were used for quantitative analysis?
Expression of selected genes -Authors should add a general comment on the biological relevance of the transcriptional data in the context of transcript -translated protein -its function in the cell, e.g. is the level of transcript directly related to the level and/or activity of the appropriate protein?What concentartion of PAL or PAA was used to obtain data presented in Fig. 3 and Table 4? Since Fig. 3 and Table 4 present data for just one concentration point thus the statement is not justified: 'The results of the present study show that PAL and PAA significantly increase the gene expression of P53 at high concentrations, and the increase in P53 gene expression is concentration dependent; the higher the concentration, the higher the level of gene expression.'Furthermore, how quantification of the PCR product, i.e. bands intensity was performed?What software was used for this analysis?Why no reference gene was used as a control for reaction efficiency?How many RT-PCR repetitions were used for quantitative analysis?
The term 'Gene expression density' should be replaced with 'Gene expression level' or 'Transcript expression level' Finally, a general comment concerning the RT-PCR technique -currently a qPCR method is considered an acceptable standard for trnascriptomic analysis.It is recommended by these reviewers to repeat analyses using this approach.
In the Introduction the rationale behind the choice of the particular genes to be analyzed (PI3K, Akt1, mTOR, P53, and EGFR) has to be presented together with a brief (one sentence) description of their function in the cancer cell.Additionally, rewrite the sentence and explain the meaning of the expression 'and differentiate between the level of individual plasmid expression in multivalent pDNA'.How this expression is relevant to the method used in this study?
Methodology requires clarification: Delete the description of the cleaning of the leaves from 'The simplicia procedure …' to '…stored in tightly closed plastic containers.'Instead, describe the date of the collection of the leaves, for how long the plant material was stored prior to extraction, the temperature of the extraction procedure.

1.
What is meant in the sentence: 'The portions were further saponified and ……..' All the ester have already been saponified upon KOH treatment.

2.
Was a crude unsaponifiable lipid fraction used for further tests?It has to be clearly mentioned in the text.

3.
If so what was the approximate polyisoprenoid content in this fraction?4.
Data obtained in the biological test are not fully justified keeping in mind the complexity of the lipid fraction used.

5.
A type of the TLC plates should be mentioned in the text, moreover what is the meaning of the expression: '… were confirmed to belong to the dolichol family 100%...'.

6.
What type of cell line are Vero cells?Which organ is it derived from?Is it an immortalized cell line?

7.
Concentration of 5-Fu used in this study -please provide literature data on the concentration of 5-FU used.Were the values used in this study within the range of those used in similar type of assays?

8.
The method of administration of the leaf extracts to the cells is not described at all.Polyisoprenoids are not soluble in water so what type of solvent was used to prepare PAA and PAL solutions for all biological tests?And how the control cells were treated -there is no information, neither in the Methods nor in the Figure legends, on the supplementation of the growth media of the control cell cultures with the appropriate amount of the same solvent.

9.
For how long the cells were exposed to PAL in the cell cycle analysis?What concentrations of PAL and PAA were used?10.
The description of the apoptosis analysis is unclear and requires rewriting.What type of software was used to count green and red fluorescence signals?

11.
The amount of total RNA used in this study in the reverse transcription reaction (according to the manuscript 3 mg in the 20 µl reaction mixture) seems unrealistic.Manufacturer's protocols usually recommend to use ≤1 µg RNA.Please correct.

12.
The novelty of the data presented in this study has to be indicated by Authors in the section Discussion or Conclusions -novel data presented in the current manuscript in comparison to the previous already published ones should be clearly depicted.
The manuscript requires an English language edit.Some sentences are misleading, some are just awkward, e.g.'Cytotoxic activity against WiDr cells showed that the IC50 for A. alba and A. lanata was 258.14 ug/mL and 243.32 ug/mL, respectively.This indicated that their classification as anticancer agents was moderate.''Natural ingredients developed as potential chemotherapeutic agents include mangrove leaves.' should rather read as follows: 'Natural substances developed as potential chemotherapeutic agents include components of mangrove leaves.' 'This extract has a mechanism for inhibiting the cell cycle at the G0-G1 phase…' 'In this study, the test material is cytotoxic for the polyisoprenoids of leaves derived from…' The word 'simplicia' is used in the text several times-please replace with any typical expression.

Minor remarks:
Title -'using reverse transcription-PCR' -in my opinion these last four words are not necessary and should be deleted; it is nothing special in this RT-PCR technique.

○
p.1 -'Mangrove plants produce a polyisoprenoid compound.Polyisoprenoids have been proven to have anticancer properties.' -these two sentences have to be rewritten.As it is now they do not provide enough information about the subject of study.Moreover, why the term 'polyisoprenoid compound' is used here in a singular form?It is not consistent either with the natural composition of the leaf extract or with the text below.This manuscript describes elucidation of the biological activity of two polyisoprenoidcontaining extracts isolated from leaves of two mangrove plant species Avicennia lanata and Avicennia alba.To meet the generally acceptable standard of the manuscript quality this preliminary study requires extremely extensive revision.
Response: We would like to thank the Reviewers' comments and suggestions which significantly improve the manuscript and enrich the content.
Firstly, all the biological data were obtained using crude and poorly characterized extracts and consequently all these experiments should be performed once again with highly purified polyisoprenoid mixture (polyisoprenoid content at least 90%).Such purification procedure is doable within a reasonable time period and is not very difficult.
Response: We agreed with Reviewers' suggestions to revise the procedures.The polyisoprenoid extraction was performed using the established procedures.The lipid extracts of Avicennia alba and A. lanata was concentrated to dryness and saponified at 65°C for 24 h in 86% ethanol containing KOH 2 M. The unsaponifiable lipid partitioned into hexane by vigorous mixing was analyzed by silica gel 60 TLC and RP-18 high performance thin layer chromatography (HPTLC) plates.The unsanifiable lipid basically denotes simple lipid fractions except for fatty acids (saponifiable lipid).The leaf extracts (50-100 mg) were applied to TLC plate.The quantity of polysioprenoids in A. alba leaves was 5.5±0.8mg/g dry weight and A. lanata leaves was 14.9±1.2mg/g dry weight.Data are represented as the means ± SEM (n=3).We enclose two-dimensional TLC (2D TLC) chromatograms. Figure 1 shows 2D TLC chromatograms of polyisoprenoids from leaves of A. alba and A. lanata.Dolichols with the chain length of C60-C100 and C70-C100 were detected as major polyisoprenoids alcohols in A. alba leaves (A) and A. lanata leaves (B), respectively.No polyprenol was found in both mangrove leaves.The 2D TLC has been performed triplicates and showed an identical pattern.We confirmed that leaves extracts contained 100% dolichols as highly purified polyisoprenoid mixture to meet Reviewers' requirement.Therefore it is not needed to purify the polyisoprenoid samples and used for further investigation.
New and revised sentences have added to the revised manuscript to incorporate Reviewers' suggestions.Please refer to the revised version of Preparation of isolation polyisoprenoid alcohols.Thirdly, interpretation of the obtained results seems not really clear, in particular results of the analysis of the toxicity should be reconsidered (see below).
Response: To incorporate Reviewers'the results of the analysis of the toxicity has been revised by adding the standard errors of the means and statistical analysis.Please refer to revised Table 2.
Finally, the Authors should carefully discuss the available literature data to document the novelty of their study.
Response: We agreed with Reviewers' suggestions, new sentences have been added to meet Reviewers' comments, please refer to revised Discussion.
A detailed list of comments follows.This manuscript describes study on the biological effects of the polyisoprenoid-containing extracts isolated from leaves Avicennia lanata and Avicennia alba.All tests were performed for two cell lines, WiDr and Vero.Shown are data on the effect of PAA and PAL on the cytotoxic activity, cell-cycle progression and expression of the selected genes: PI3K, Akt1, mTOR, and EGFR.General comments: All the biological tests described in this study were performed using the crude mixture of lipids obtained after alkaline hydrolysis of the leaf extracts.The Authors did not provide the approximate polyisoprenoid content in the mixtures used.Such complex mixture of natural compounds could be used just for preliminary tests of their potential biological activity.
Response: Unsaponifiable lipid was analysed using TLC and 2D-TLC plates to identify polyisoprenoids that contained 100% dolichols with the chain length of C60-C100 and C70-C100 detected as main polyisoprenoids alcohols in A. alba leaves and A. lanata leaves.New sentences have been added to the revised manuscript.Please refer to revised Preparation of isolation polyisoprenoid alcohols.
What is more, data presented in this study in fact confirm the weakness of the strategy used -e.g.different effects of pro-apoptocic activity of PAA and PAL on the level of expression of analyzed genes might suggest that this is NOT the effect of polyisoprenoids (very similar profile for both extracts) but rather some other components of these mixtures.
Response: We clarified that leaves extracts of PAA and PAL contained 100% dolichols based on the HPTLC chromatograms (Figure 1), we believe that different effects of pro-apoptotic activity of PAL and PAL on the level of expression of analysed from the effect of refer to revised part Results of Discussion of Expression of PI3K, Akt1, mTOR, P53, and EGFR genes Finally, a general comment concerning the RT-PCR technique -currently a qPCR method is considered an acceptable standard for trnascriptomic analysis.It is recommended by these Reviewers to repeat analyses using this approach.
Response: We agreed with Reviewers' suggestion, however, in the present situation, the authors are unable to repeat analysis using transcriptomic approach.
In the Introduction the rationale behind the choice of the particular genes to be analyzed (PI3K, Akt1, mTOR, P53, and EGFR) has to be presented together with a brief (one sentence) 1.
Response: The description of the cleaning of the leaves from 'The simplicia procedure …' to '…stored in tightly closed plastic containers.' has been deleted from revised manuscript.What is meant in the sentence: 'The portions were further saponified and ……..' All the ester have already been saponified upon KOH treatment.

1.
Response: The sentence: 'The portions were further saponified and ……..' has been deleted and changed to new sentence to read 'The unsaponifiable lipid partitioned into 2 mg/mL nhexane'.Was a crude unsaponifiable lipid fraction used for further tests?It has to be clearly mentioned in the text.

1.
Response: A crude unsaponifiable lipid used for further test and has been clearly mentioned in the text of revised manuscript.
If so what was the approximate polyisoprenoid content in this fraction?1.
The quantity of polysioprenoids in A. alba leaves was 5.5±0.8mg/g dry weight and A. lanata leaves was 14.9±1.2mg/g dry weight.Data are represented as the means ± SEM (n=3).
Data obtained in the biological test are not fully justified keeping in mind the complexity of the lipid fraction used.

1.
Response: We agreed with Reviewers' comments on the complexity of the lipid fraction, however, the biological tests have been clarified to be 100% dolichols.
A type of the TLC plates should be mentioned in the text, moreover what is the meaning of the expression: '… were confirmed to belong to the dolichol family 100%...'.

1.
Response: Types of TLC plates were Silica gel 60 thin layer chromatography (TLC) and RP-18 HPTLC plates have been added to revised Methods, the expression: '….were confirmed to belong to the dolichol family 100%...' has been revised to read, '…were detected to be 100% dolichol family...' What type of cell line are Vero cells?Which organ is it derived from?Is it an immortalized cell line? 1.
Response: Vero ATCC® CCL-81™, an immortalized cell line, derived from the kidney of an African green monkey Concentration of 5-Fu used in this study -please provide literature data on the concentration of 5-FU used.Were the values used in this study within the range of those used in similar type of assays? 1.
Response: New sentence has been added to meet Reviewers' comment.50 μM of 5fluorouracil (5-Fu) were dissolved in a 100 μL dimethyl sulfoxide (DMSO) co-solvent.The concentration used in this study within the rage of those used in colon cancer cells.
The method of administration of the leaf extracts to the cells is not described at all.Polyisoprenoids are not soluble in water so what type of solvent was used to prepare PAA and PAL solutions for all biological tests?And how the control cells were treated -there is no information, neither in the Methods nor in the Figure legends, on the supplementation of the growth media of the control cell cultures with the appropriate amount of the same solvent.

1.
Response: To incorporate Reviewers' comments, new sub title in the Methods have been added.Please refer to Administration of the leaf extracts to the cells of Methods.
For how long the cells were exposed to PAL in the cell cycle analysis?What concentrations of PAL and PAA were used? 1.
Response: The cell was exposed to serially diluted concentrations of PAA and PAL (1000, 500, 250, 125, and 62.5 µg/mL) in the cell cycle analysis for 48 h.Please refer to revised sentence in the Cytotoxicity analysis of Methods.The description of the apoptosis analysis is unclear and requires rewriting.What type of software was used to count green and red fluorescence signals? 1.
Response: New sentence has been added to meet Reviewers' comments.ImageRaster 4.05.(Micronos, Yogyakarta, Indonesia) was used to count green and red fluorescence signals.
Please refer to revised Apoptosis analysis method.The amount of total RNA used in this study in the reverse transcription reaction (according to the manuscript 3 mg in the 20 µl reaction mixture) seems unrealistic.Manufacturer's protocols usually recommend to use ≤1 µg RNA.Please correct.

1.
Response: The total RNA (0.3 µg each) has been corrected.The novelty of the data presented in this study has to be indicated by Authors in the section Discussion or Conclusions -novel data presented in the current manuscript in comparison to the previous already published ones should be clearly depicted.
Response: We agreed with Reviewers' suggestion, new and revised sentences have added to revised Conclusion.The manuscript requires an English language edit.Some sentences are misleading, some are just awkward, e.g.'Cytotoxic activity against WiDr cells showed that the IC50 for A. alba and A. lanata was 258.14 ug/mL and 243.32 ug/mL, respectively.This indicated that their classification as anticancer agents was moderate.'Response: To incorporate Reviewers' comments, the sentences have been revised to read 'Cytotoxic activity against WiDr cells showed that the IC50 for A. alba and A. lanata was 258.14 ug/mL and 243.32 ug/mL, respectively.This observation indicated the possibility to develop moderate anticancer agents.' 'Natural ingredients developed as potential chemotherapeutic agents include mangrove leaves.' should rather read as follows: 'Natural substances developed as potential chemotherapeutic agents include components of mangrove leaves.'Response: Natural ingredients developed as potential chemotherapeutic agents include mangrove leaves.' has been corrected to 'Natural substances developed as potential chemotherapeutic agents include components of mangrove leaves.' 'This extract has a mechanism for inhibiting the cell cycle at the G0-G1 phase…' Response: 'This extract has a mechanism for inhibiting the cell cycle at the G0-G1 phase…' has been revised to 'Polyisoprenoids have a mechanism to inhibit the cell cycle at the G0-G1 phase…' 'In this study, the test material is cytotoxic for the polyisoprenoids of leaves derived from…' Response: 'In this study, the test material is cytotoxic for the polyisoprenoids of leaves derived from…'has been corrected to 'In this study, the cytotoxic test material was derived from polyisoprenoids of mangrove leaves (PAA and PAL)..' The word 'simplicia' is used in the text several times-please replace with any typical expression.
The word 'simplicia' has been deleted to incorporate Reviewers' suggestion throughout the revised manuscript.
Minor remarks: Title -'using reverse transcription-PCR' -in my opinion these last four words are not necessary and should be deleted; it is nothing special in this RT-PCR technique.
○ Response: 'using reverse transcription-PCR' has been deleted from the title.p.1 -'Mangrove plants produce a polyisoprenoid compound.Polyisoprenoids have been proven to have anticancer properties.' -these two sentences have to be rewritten.As it is now they do not provide enough information about the subject of study.Moreover, why the term 'polyisoprenoid compound' is used here in a singular form?It is not consistent either with the natural composition of the leaf extract or with the text below.Response: '…dolichol and polyprenol on the leaves…' has been replaced with '…dolichol and polyprenol in the leaves…'.

Figure 1 .
Figure 1.Cell cycle analysis showing the percentage accumulated in each phase of the cell cycle WiDr after the administration of the extract of mangrove leaves and 5-Fu as a positive control: (a) Control cell; (b) PAL 1/5 IC 50 ; (c) PAA1/5IC 50 ; and (d) 5-Fu1/5 IC 50 .
cancer cells undergo apoptosis and lose their ability to proliferate rapidly.This way of treatment may induce usual apoptotic signalling, thereby, potentially eliminating the cancer cells 26 .The potential working mechanism of PAL and PAA are in the late phase of apoptosis.The potency of PAL and PAA in triggering apoptosis may be caused by compound isoprenoids (based on the results of phytochemical screening of PAL and PAA).Steroids/triterpenoids are compounds that have high anticancer activity, by blocking nuclear factorkappa B, inducing apoptosis, and activating transcription and angiogenesis, which can be useful in the treatment of various types of cancer 27 .

and Figure 3 .
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Table 4 .
Gene expression of n-hexane extract of mangrove leaves Avicennia alba (PAA) and Avicennia lanata (PAL) in WiDr cells.No Gene Treatment group Density average value ± 0,01 abc a. Sig (P) < 0.05: There is a significant difference with the normal group (control cell).

○ p. 1 -
delete 'inhibited' in the sentence 'The inhibited cell cycle and apoptosis...'.○ p.3 -rewrite the second sentence of Introduction -what is meant by the expression 'halt its invasion and metastasis' in the context of this sentence?○ p.3 -replace '…dolichol and polyprenol on the leaves…' with '…dolichol and polyprenol in the leaves…'.○p.3 -'MCF-7 and T47D' -a brief description of the cell line type is missing.○ p.3 -'gene expression of COX-2 in colon cancer cells' spell out the name of the enzyme ○

○'
Mangrove plants produce polyisoprenoid compounds.Polyisoprenoids have been proven to have anticancer properties.' p.1 -delete 'inhibited' in the sentence 'The inhibited cell cycle and apoptosis...'.○ Response: 'inhibited' has been deleted.p.3 -rewrite the second sentence of Introduction -what is meant by the expression 'halt its invasion and metastasis' in the context of this sentence?○ Response: The second sentence of Introduction 'halt its invasion and metastasis' has been corrected to 'spread its invasion and metastasis' in the context of this sentence.p.3 -replace '…dolichol and polyprenol on the leaves…' with '…dolichol and polyprenol in the leaves…'.

Table 3 . Accumulated values in each phase of the cell cycle of WiDr cells treated with of mangrove leaves Avicennia alba (PAA) and Avicennia lanata (PAL). Treatment Concentrations (μg/mL) Phase of the cell cycle (%) G0-G1 S G2-M
description of their function in the cancer cell.Response: To incorporate Reviewers' suggestion, a new sentences relating to description function of PI3K, Akt1, mTOR, p53, and EGFR in the cancer cell has been added to Introduction.Please refer to last sentence of Introduction.Additionally, rewrite the sentence and explain the meaning of the expression 'and differentiate between the level of individual plasmid expression in multivalent pDNA'.How this expression is relevant to the method used in this study?Response: The sentence 'and differentiate between the level of individual plasmid expression in multivalent pDNA' has been deleted from revised manuscript.Methodology requires clarification:Delete the description of the cleaning of the leaves from 'The simplicia procedure …' to '…stored in tightly closed plastic containers.'Instead, describe the date of the collection of the leaves, for how long the plant material was stored prior to extraction, the temperature of the extraction procedure.