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. 2024 Apr 17;19(4):e0299920. doi: 10.1371/journal.pone.0299920

Jolkinolide B inhibits the progression of hepatocellular carcinoma by regulating Musashi-2 protein

Tianchun Wu 1,2,*, Han Yang 1,2, Jinjin Li 1,2, Hongbo Fang 1,2, Xiaoyi Shi 1,2, Jie Li 1,2, Liushun Feng 1,2
Editor: Kishor Pant3
PMCID: PMC11023458  PMID: 38630658

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. However, the HCC treatment is still challenging. Herein, we aimed to reveal the anti-tumor effect of Jolkinolide B in HCC cell lines Huh-7 and SK-Hep-1. The results showed that Jolkinolide B inhibited the migration, invasion, and epithelial-to-mesenchymal transition(EMT) of HCC cells. In addition, Jolkinolide B induced HCC cell apoptosis by upregulating Bax and downregulating BCL-2 expressions. Furthermore, we demonstrated that Jolkinolide B inactivated the β-catenin signaling and reduced Musashi-2 expression. Finally, we revealed that Musashi-2 overexpression reversed the Jolkinolide B-induced anti-HCC effect. Overall, we proved that Jolkinolide B is a potential candidate for treating HCC.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors [1]. Although the survival rate of patients with HCC has improved, HCC is the fifth leading causes of cancer-related deaths worldwide, and effective therapeutic approaches for HCC in clinical treatment are still lacking [2, 3]. Therefore, developing efficient pharmaceutical agents against HCC is urgently necessary.

Musashi-2 (MSI2), a member of the MSI family, is a conserved RNA-binding protein [4, 5]. MSI2 is an important regulation factor of sperm and embryo formation. Currently, numerous studies have shown that MSI2 plays a crucial role in mediating cancer stemness, migration, invasion, proliferation, and EMT. In addition, MSI2 can be a prognostic factor for tumors [6–8]. For instance, MSI2 has been identified as a prognostic biomarker for gastric, cervical, and non-small cell lung cancers [9–11]. Moreover, MSI2 promotes bladder cancer migration and invasion by activating the JAK2/STAT3 signaling pathway [6].

Jolkinolide B, extracted from the traditional Chinese herb Euphorbia fischeriana Steud., is a bioactive diterpenoid with anti-tumor effects against several tumors [12]. For example, Jolkinolide B induces gastric cancer cell apoptosis and cell cycle in vitro, and inhibits tumor growth in vivo. In addition, Jolkinolide B induces MCF-7 cell apoptosis by inactivating the PI3K/Akt/mTOR signaling pathway [13, 14]. Jolkinolide B inhibited the breast cancer MDA-MB-231 cell line owing to its anti-metastatic effects [15]. However, the effects of Jolkinolide B on HCC remains unclear. In this study, we explored the biological effects and mechanisms of action of Jolkinolide B on HCC. This study provides a novel therapeutic approach for HCC.

Materials and methods

Cell lines and reagents

Human L-02 cells and HCC Huh-7 and SK-Hep-1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin sulfate. The cells were incubated at 37 ºC in a 5% CO2 incubator. Jolkinolide B was purchased from MedChemExpress and dissolved in dimethyl sulfoxide (DMSO).

Cell viability assay

The antiproliferative effect of Jolkinolide B was evaluated in L-02, Huh-7 and SK-Hep-1 cell lines. Briefly, 2 ×103 cells were seeded in 96-well plates and cultured overnight. In the following day, cells were treated with different concentrations of Jolkinolide B (0, 5, 10, 25, 50, or 100 μM) for 48 h. Finally, 10 μL Cell Counting Kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Japan) was added to each well. The cells were then cultured for 2 h. The optical density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Lentivirus-mediated MSI2 overexpression

Huh-7 and SK-Hep-1 cells were seeded in six-well plates and incubated overnight at 60% confluence. Lentivirus-mediated MSI2 plasmids were added to HCC cells and incubated for 48 h. Then, the total protein and RNA were extracted from the cells for subsequent experiments, as described in the following sections.

Western blotting

Cells were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific Inc.) containing phenylmethylsulfonyl fluoride and phosphatase inhibitors. The proteins were quantified using bicinchoninic acid protein assay. Proteins were then separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk at room temperature (20–25 ºC) for 1 h and incubated with primary antibodies at 4 ºC overnight. After incubation, the membranes were washed thrice with 1 × tris-buffered saline with 0.1% Tween® 20 for 10 min and incubated with secondary antibodies for 1 h at room temperature. Antibodies against MSI2 (ab76148), Bax (ab32503), and BCL-2 (ab32124) were purchased from Abcam (Cambridge, UK), and antibodies against E-cadherin (#14472), vimentin (#5741), GAPDH (#97166), p53 (#9282) and β-catenin (#8480) were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Flow cytometry analysis

Jolkinolide B-induced HCC cell apoptosis was evaluated using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). Briefly, Huh-7 and SK-Hep-1 cells were cultured in six-well plates and treated with Jolkinolide B. Huh-7 and SK-Hep-1 (1 × 106 cells) cell suspension were collected, washed three times with pre-cold phosphate-buffered saline buffer, and stained with 5 μL Annexin V-FITC and 4 μL propidium iodide in the dark for 30 min. The cell apoptosis was analyzed using a Flow Cytometer (BD Biosciences, San Jose, CA, USA). Finally, the HCC apoptotic cell rates were calculated.

Real-time quantitative polymerase chain reaction

Total RNA was extracted using a TRIzol kit (Invitrogen, Thermo Fisher Scientific, Inc.). The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The RNA was transcribed into cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, China). The SYBR Green PCR Master Mix method was used to amplify the target genes. The 2−ΔΔCt method was used to analyze the relative expression of the target genes. GAPDH was used as an internal control. The primers used in this study were: MSI2, 5’-ATCCCACTACGAAACGCTCC-3’ (forward) and 5’-GGGGTCAATCGTCTTGGAATC-3’ (reverse); matrix metalloproteinase-7 (MMP-7), 5’-GAGTGAGCTACAGTGGGAACA-3’ (forward) and 5’- CTATGACGCGGGAGTTTAACAT-3’ (reverse); c-Myc, 5’-GGCTCCTGGCAAAAGGTCA-3’ (forward) and 5’-CTGCGTAGTTGTGCTGATGT-3’ (reverse); and GAPDH, 5’-CTGGGCTACACTGAGCACC-3’ (forward) and 5’- AAGTGGTCGTTGAGGGCAATG-3’ (reverse).

Wound healing assay

Huh-7 or SK-Hep-1 cells were seeded in six-well plates overnight and scratched with a 100-μL pipette tip. The scratched wound observed under an inverted microscope, and images were collected. After incubation with DMSO or Jolkinolide B at a concentration of 10 μM for 48 h, the migrated area was photographed.

Transwell assay

Huh-7 or SK-Hep-1 cells were seeded into the upper chamber of transwell plates in 500 μL DMEM containing DMSO or Jolkinolide B. The lower chamber contained DMEM supplemented with 20% fetal bovine serum. After incubating for 48 h, the cells in the lower chamber were fixed with paraformaldehyde for 1 h and stained with 0.1% crystal violet. Then, the cells were counted under an optic microscope.

Statistical analysis

Cell experiments were repeated in triplicate. Data were expressed as mean values ± standard deviation. One-way analysis of variance was performed for multiple comparisons among different groups. Data processing and analysis were performed using GraphPad Prism software (La Jolla, CA, USA). A p-value <0.05 was considered statistically significant, and *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001.

Results

Jolkinolide B reduces HCC cell lines viability

To identify the anti-tumor effects of Jolkinolide B on HCC cells, Huh-7 and SK-Hep-1 cells were treated with different concentrations (0, 5, 10, 25, 50, or 100 μM) of Jolkinolide B for 48 h and the cell viability was assessed using CCK-8 assay. IC50 for Huh-7 and SK-Hep-1 cells were 14.09 μM and 11.17 μM, respectively. The results showed that Jolkinolide B decreased HCC cell viability in a dose-dependent manner. Furthermore, we found that Jolkinolide B did not cause cytotoxicity to L-02 cells (Fig 1). Together, these results indicate that Jolkinolide B inhibits hepatocellular carcinoma cells.

Fig 1. Jolkinolide B inhibits HCC cells proliferation.

Fig 1

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Cell viability assays of Huh-7 (A), SK-Hep-1 (B) and L-02 cells (C) were performed using CCK-8 method after treatment with different concentrations (0, 5, 10, 25, 50, or 100 μM) of Jolkinolide B for 48 h.

Jolkinolide B inhibits HCC cell migration and promotes HCC cell apoptosis

To investigate the effects of Jolkinolide B on HCC cell migration, invasion, and EMT, we performed wound healing assay using Huh-7 and SK-Hep-1 cells. Transwell assay was performed to estimate the anti-tumor effects of Jolkinolide B on the migration and invasion of HCC cells. The results showed that Jolkinolide B inhibited HCC cell migration and invasion (Fig 2A and 2B). Furthermore, we performed western blotting to estimate the effect of Jolkinolide B on EMT. The results showed that Jolkinolide B upregulated the EMT marker E-cadherin and downregulated vimentin protein expression (Fig 2C). Apoptosis plays an important role in traditional Chinese herbal-induced anti-tumor effect. Therefore, we investigated the effect of Jolkinolide B on HCC cell apoptosis using western blotting to evaluate Bax and BCL-2 protein expressions. The results showed that Jolkinolide B increased Bax protein expression, whereas it decreased BCL-2 protein levels (Fig 2D). Besides, we applied flow cytometry to analyze the apoptosis rates of HCC cells induced by Jolkinolide B, the results showed that Jolkinolide B could induce HCC cells apoptosis (Fig 2E). Collectively, Jolkinolide B inhibited HCC cell migration, invasion, and EMT, and induced cell apoptosis.

Fig 2. Jolkinolide B inhibits HCC cell lines migration, invasion and promotes HCC cell apoptosis.

Fig 2

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(A) Huh-7 and SK-Hep-1 cells were seeded, scratched, and then treated with Jolkinolide B at a concentration of 10 μM for 48 h. The wound healing assays were performed to assess the migration abilities of Huh-7 and SK-Hep-1 cells. (B) Huh-7 and SK-Hep-1 cells were seeded in the upper transwell chamber and treated with DMSO or Jolkinolide B of 10 μM for 48 h to evaluate the migration and invasion abilities of HCC cells. (C) Huh-7 and SK-Hep-1 cells were treated with DMSO or Jolkinolide B at a concentration of 10 μM for 48 h; then the protein expression of E-cadherin and vimentin was analyzed using western blotting. GAPDH was used as an internal control. (D) Western blotting was used to assess Bax and BCL-2 protein expressions. GAPDH was used as an internal control. The relative protein intensities were analyzed. (E) Huh-7 and SK-Hep-1 cells were treated with DMSO or Jolkinolide B, the cells apoptosis was analyzed by flow cytometry and apoptosis rates were calculated. *p <0.05, **p <0.01 and ***p <0.001.

Jolkinolide B inactivates β-catenin signaling pathway

The activation of the β-catenin signaling pathway promotes tumor progression. Therefore, the inactivation of the β-catenin signaling pathway is an effective method for inhibiting tumor progression. To investigate the effect of Jolkinolide B on β-catenin signaling pathway, Huh-7 (Fig 3A) and SK-Hep-1 (Fig 3B) cells were treated with different concentrations of Jolkinolide B (0, 5, 10, 25, 50, or 100 μM). The results showed that Jolkinolide B downregulated β-catenin protein expression in a dose-dependent manner. In addition, the results showed that the downstream genes of β-catenin signaling MMP-7 and C-MYC were also downregulated by Jolkinolide B treatment (Fig 3C and 3D).

Fig 3. The effects of Jolkinolide B on the β-catenin pathway in HCC cells.

Fig 3

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(A) Huh-7 and (B) SK-Hep-1 cells were treated with the indicated concentrations of Jolkinolide B for 48 h. The β-catenin protein expression was analyzed using western blotting. GAPDH was used as an internal control. The relative protein intensities were analyzed. (C, D) Huh-7 and SK-Hep-1 cells were treated with 10 μM Jolkinolide B for 48 h. The mRNA expression of MMP-7 and C-MYC in Huh-7 (C) and SK-Hep-1 (D) cells analyzed using RT-qPCR method. GAPDH was used as an internal control. *p <0.05, **p <0.01, ***p <0.001 and ****p <0.0001.

Jolkinolide B downregulates MSI2 expression

Previous studies have reported that MSI2 is an important marker of tumor progression and is vital for tumor metastasis, proliferation, and cell cycle in different types of tumors, including HCC. To investigate whether Jolkinolide B can induce the expression of MSI2, Huh-7 and SK-Hep-1 cells were treated with different concentrations of Jolkinolide B (0, 5, 10, 25, 50, or 100 μM). The results showed that Jolkinolide B downregulated the MSI2 protein expression in Huh-7 (Fig 4A) and SK-Hep-1 (Fig 4B) cells. p53 shows as an important cancer suppressor, we detected whether Jolkinolide B induced p53 expression, western blots showed that Jolkinolide B could upregulate p53 expression (Fig 4C and 4D).

Fig 4. Jolkinolide B downregulates the protein of MSI2 and the p53 expression in HCC cells.

Fig 4

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(A, C) Huh-7 and (B, D) SK-Hep-1 cells were treated with the indicated concentrations of Jolkinolide B for 48 h. The MSI2 and p53 protein expressions were analyzed using western blotting. GAPDH was used as an internal control. *p <0.05, **p <0.01 and ***p <0.001.

MSI2 overexpression promotes β-catenin signaling

To identify the role of MSI2 on the regulation of β-catenin signaling, this study overexpressed MSI2 expression in both Huh-7 and SK-Hep-1 cells (Fig 5A and 5B). β-catenin was detected after MSI2 overexpression (Fig 5C). These results indicated that MSI2 overexpression promoted β-catenin expression.

Fig 5. MSI2 overexpression promotes β-catenin expression in HCC cells.

Fig 5

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Huh-7 and SK-Hep-1 cells were transfected with lentivirus-mediated MSI2 overexpression plasmids for 48 h, the MSI2 protein expression (A) and MSI2 mRNA expression (B) were analyzed using western blotting and RT-qPCR methods, respectively. (C) β-catenin protein was detected in Huh-7 and SK-Hep-1 cells after MSI2 overexpression. The relative protein intensities were analyzed. *p <0.05, **p <0.01 and ***p <0.001.

MSI2 overexpression reverses Jolkinolide B-induced inhibition of HCC cells

The above results showed that Jolkinolide B inhibited HCC cell migration, induced apoptosis, and downregulated MSI2 expression. To further investigate the effect of MSI2 in Jolkinolide B-induced HCC progression, Huh-7 and SK-Hep-1 cells were treated with DMSO, Jolkinolide B, or Jolkinolide B and MSI2 plasmids. The results showed that MSI2 reversed Jolkinolide B-induced upregulation of Bax and downregulation of BCL-2 (Fig 6A). Therefore, we further investigated the effect of MSI2 in Jolkinolide B-induced EMT. The results demonstrated that MSI2 reversed Jolkinolide B-induced upregulation of E-cadherin and downregulation of vimentin (Fig 6B). Finally, β-catenin protein expression in Huh-7 and SK-Hep-1 cells was also reversed by MSI2 overexpression in Jolkinolide B-induced downregulation of β-catenin signaling (Fig 6C).

Fig 6. Overexpression of MSI2 reverses Jolkinolide B-promoted apoptosis and Jolkinolide B-inhibited EMT and β-catenin signaling in HCC cells.

Fig 6

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Huh-7 and SK-Hep-1 cells were treated with DMSO, 10 μM Jolkinolide B, or Jolkinolide B together with MSI2 plasmids. Then, the protein expressions of (A) Bax, BCL-2, (B) E-cadherin, vimentin and (C) β-catenin were analyzed using western blotting. GAPDH was used as an internal control. The relative protein intensities were analyzed. (D) Proposed working model of Jolkinolide B-induced inhibition of HCC. *p <0.05, **p <0.01 and ***p <0.001.

Discussion

Recent studies have shown that Jolkinolide B exerts anti-tumor activities against many tumors by regulating tumor metastasis and apoptosis through different mechanisms [13]. During tumor metastasis, epithelial cells lose polarity, which is acquired by mesenchymal cells, contributing to tumor cell migration and invasion [16–18]. In this study, we demonstrated that Jolkinolide B reduced the migration and invasion of Huh-7 and SK-Hep-1 cells. E-cadherin is a biomarker of epithelial cells, whereas vimentin is a biomarker of mesenchymal cells. Therefore, EMT is characterized by the loss of E-cadherin and acquisition of vimentin [19–21]. In this study, Jolkinolide B inhibited EMT progression, resulting in an increase in E-cadherin and a decrease in vimentin levels. Cellular apoptosis is programmed cell death. The Bax/BCL-2 ratio changes play an important role in mediating the downstream apoptotic cascades that leads to apoptosis [22, 23]. Herein, we demonstrated through western blot analysis that Jolkinolide B increased Bax protein levels and decreased BCL-2 protein expression. Our results showed that Jolkinolide B inhibits HCC cell migration and induces apoptosis.

Several studies showed that Wnt/β-catenin plays an important role in mediating tumor cell apoptosis and migration. Therefore, the inactivation of Wnt/β-catenin signaling pathway has been proposed as a potential therapeutic target [24–26]. For instance, paeonol exerts anti-colorectal cancer effects by inhibiting the Wnt/β-catenin signaling, whereas baicalin inhibits highly aggressive breast cancer by targeting Wnt/β-catenin signaling [27, 28]. In our study, we revealed that Jolkinolide B inhibits the β-catenin signaling by reducing the β-catenin protein expression.

MSI2, which is highly expressed in the hematopoietic system, contributes to hematopoietic stem cell regulation, and sperm and embryo formation [29, 30]. Recent studies have shown that MSI2 is closely related to tumor cell proliferation, invasion, migration, and apoptosis, and is an important regulator of various signaling pathways [6, 10, 31]. In addition, MSI2 is critical for HCC progression; for instance, MSI2 contributes to HCC cell stemness and chemoresistance, and MSI2 promotes hepatitis B virus-related HCC [7, 32]. Therefore, targeting MSI2 may be an effective approach for treating HCC. In this study, we used Jolkinolide B to target MSI2 and found that Jolkinolide B reduced MSI2 expression.

In summary, this study found that Jolkinolide B inhibited HCC by promoting apoptosis, reducing cell migration, and inducing β-catenin signaling inactivation. In addition, we further discovered that Jolkinolide B reduces the MSI2 expression, which can reverse Jolkinolide B-induced anti-tumor effects (Fig 6D). This study provides a novel therapeutic target for treating HCC.

Supporting information

S1 File

Slide 1: Original images for Fig 2A, 2B, 2E. Slide 2: Original immunoblots for Fig 2C, 2D. Slide 3: Original immunoblots for Fig 3. Slide 4: Original immunoblots for Fig 4. Slide 5: Original immunoblots for Fig 5A, 5C. Slide 6: Original immunoblots for Fig 6A, 6B. Slide 7: Original immunoblots for Fig 6C. Sheet 1: Original CCK8 of cell viability for Fig 1A-1C. Original RT-qPCR for Figs 3C, 3D, 5B.

(ZIP)

pone.0299920.s001.zip (3.4MB, zip)

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

Medical scientific and technological research joint construction projects of Henan province,2020. NO.LHGJ20200361.

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Decision Letter 0

Kishor Pant

11 Dec 2023

PONE-D-23-36751Jolkinolide B inhibits the progression of hepatocellular carcinoma through regulating musashi-2 proteinPLOS ONE

Dear Dr. Wu

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Kishor Pant

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Based on the information presented in the study, the following concerns are proposed as prospective enhancements:

Major revision.

The manuscript represents the role of Jolkinolide B in the progression of hepatocellular carcinoma through the regulation of Musashi-2 protein; however, the authors did not investigate the mechanism in detail. They may detect changes in the survival signaling pathways such as STAT3, p53, AP-1, ATF6, or PI3K/AKT pathways.

Minor revision.

1. The manuscript is standard English, though some errors should be rectified.

2. In the legends of Figures 3 and 4, gapdh should be GAPDH, and also in materials and method of RT-qPCR.

3. Project No. in the funding section needs to be included.

Reviewer #2: The manuscript entitled “Jolkinolide B inhibits the progression of hepatocellular carcinoma through regulating musashi-2 protein” provides new insights that, Jolkinolide B as a potential candidate against hepatocellular carcinoma. It needs major revision to make the suitable for publication.

1. IC50 value of Jolkinolide B should be calculated.

2. Authors should confirm the apoptosis induced by Jolkinolide B, Annexin V or Tunnel assay in both lines.

3. In figure 2 legend, authors should mention concentration of JB and time of treatment of better understanding.

4. Similarly in Figure 3 and 4 legend, mention the treatment time.

5. Authors also should include the effect of JB on normal hepto cell lines.

6. All Western blots in manuscript should be quantified.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: PONE-D-23-36751_comments.docx

pone.0299920.s002.docx (13.8KB, docx)
PLoS One. 2024 Apr 17;19(4):e0299920. doi: 10.1371/journal.pone.0299920.r002

Author response to Decision Letter 0

3 Feb 2024

Dear Editor and Reviewer:

Thank you for your letter and for the reviewer’s comments concerning our manuscript entitled “Jolkinolide B inhibits the progression of hepatocellular carcinoma by regulating Musashi-2 protein”. We very much appreciate all of the comments received in the review. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researchers. We have studied these comments carefully and have made corrections accordingly. Our responses are given in a point-by-point manner below. Changes to the manuscript are shown with track changes.

We hope the revised version is now suitable for publication and look forward to hearing from you in due course.

Responds to the reviewer’s comments:

Reviewer #1: Based on the information presented in the study, the following concerns are proposed as prospective enhancements:

Major revision.

The manuscript represents the role of Jolkinolide B in the progression of hepatocellular carcinoma through the regulation of Musashi-2 protein; however, the authors did not investigate the mechanism in detail. They may detect changes in the survival signaling pathways such as STAT3, p53, AP-1, ATF6, or PI3K/AKT pathways.

Reply:

We thank the reviewer’s question and following the reviewer’s suggestion, as p53 protein having an important role in inhibiting cancer progression, we have now detected the p53 protein expression in Jolkinolide B-induced HCC cell lines in Supplementary information.

Minor revision.

The manuscript is standard English, though some errors should be rectified.

Reply:

We thank the reviewer’s question. We have now reviewed and revised our manuscript with the help of native English-speakers.

In the legends of Figures 3 and 4, gapdh should be GAPDH, and also in materials and method of RT-qPCR.

Reply:

We thank the reviewer’s question. We have now revised the writings in the manuscript.

3. Project No. in the funding section needs to be included.

Reply:

We thank the reviewer’s question. The project number in the funding section has been included in the revised manuscript.

Reviewer #2: The manuscript entitled “Jolkinolide B inhibits the progression of hepatocellular carcinoma through regulating musashi-2 protein” provides new insights that, Jolkinolide B as a potential candidate against hepatocellular carcinoma. It needs major revision to make the suitable for publication.

IC50 value of Jolkinolide B should be calculated.

Reply:

We thank the reviewer’s question and the IC50 values of Jolkinolide B in HCC cell lines have been calculated.

2.Authors should confirm the apoptosis induced by Jolkinolide B, Annexin V or Tunnel assay in both lines.

Reply:

We thank the reviewer’s question. We have detected the apoptosis of HCC cell lines treated by DMSO or Jolkinolide B administration by flow cytometry.

3. In figure 2 legend, authors should mention concentration of JB and time of treatment of better understanding.

Reply:

We thank the reviewer’s question and we are sorry for having not described the experiments more clearly. We have now provided the description of Jolkinolide B concentration and time of treatment in the revised manuscript.

4. Similarly in Figure 3 and 4 legends, mention the treatment time.

Reply:

We thank the reviewer’s question and we are sorry for having not described the experiments more clearly. We have now provided more precise description of Jolkinolide B’s treatment time in the revised manuscript.

5. Authors also should include the effect of JB on normal hepto cell lines.

Reply:

We thank the reviewer’s question and we now have detected the effect of Jolkinolide B on normal hepto cell line.

6. All Western blots in manuscript should be quantified.

Reply:

We thank the reviewer’s question and following the reviewer’s suggestion, we have quantified the normalized intensity band ratios of all western blots in revised manuscript.

Attachment

Submitted filename: Response to Reviewer.docx

pone.0299920.s003.docx (17.6KB, docx)

Decision Letter 1

Kishor Pant

19 Feb 2024

Jolkinolide B inhibits the progression of hepatocellular carcinoma by regulating Musashi-2 protein

PONE-D-23-36751R1

Dear Dr. Wu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kishor Pant

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Manuscript looks good after revision and authors have made necessary changes according to reviewer's comments. So I would recommend for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

Acceptance letter

Kishor Pant

5 Apr 2024

PONE-D-23-36751R1

PLOS ONE

Dear Dr. Wu,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kishor Pant

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    Slide 1: Original images for Fig 2A, 2B, 2E. Slide 2: Original immunoblots for Fig 2C, 2D. Slide 3: Original immunoblots for Fig 3. Slide 4: Original immunoblots for Fig 4. Slide 5: Original immunoblots for Fig 5A, 5C. Slide 6: Original immunoblots for Fig 6A, 6B. Slide 7: Original immunoblots for Fig 6C. Sheet 1: Original CCK8 of cell viability for Fig 1A-1C. Original RT-qPCR for Figs 3C, 3D, 5B.

    (ZIP)

    pone.0299920.s001.zip (3.4MB, zip)
    Attachment

    Submitted filename: PONE-D-23-36751_comments.docx

    pone.0299920.s002.docx (13.8KB, docx)
    Attachment

    Submitted filename: Response to Reviewer.docx

    pone.0299920.s003.docx (17.6KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

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