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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 4  |  Page : 432-440

Ginsenoside 3β-O-Glc-DM (C3DM) enhances the antitumor activity of Taxol on Lewis lung cancer by targeting the interleukin-6/Jak2/STAT3 and interleukin-6/AKT signaling pathways


1 Department of Pharmacology, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study; Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2 Department of Biosynthesis, State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Date of Submission31-Mar-2020
Date of Acceptance10-Jun-2020
Date of Web Publication05-Oct-2020

Correspondence Address:
PhD. Yan Li
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_51_20

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  Abstract 


Objective: Nonsmall-cell lung cancer (NSCLC) is an aggressive, highly chemoresistant disease. Taxol is an effective chemotherapeutic drug widely used for the treatment of NSCLC. However, the clinical use of Taxol is limited due to the occurrence of adverse side effects under high therapeutic doses. Therefore, it is desirable to explore combination therapy to reduce the dose of chemotherapeutic drugs and achieve excellent outcomes. A biosynthetic ginsenoside, 3-O-β-D-glucopyranosyl-dammar-24-ene-3β, 20S-diol (3β-O-Glc-DM, C3DM) is obtained from microbial fermentation by metabolic engineering. Based on previous study findings, we aimed to explore the mechanism of combination therapy with C3DM and Taxol and its increasing antitumor effect on Lewis lung cancer (LLC) in this study. Materials and Methods: A thiazolyl blue tetrazolium bromide (MTT) assay was performed to evaluate cell viability; the apoptotic effect was studied using cell apoptosis assay. The Lewis tumor xenograft experiment was performed to determine the effects of C3DM combined with Taxol on tumor growth in vivo, and western blotting was performed to analyze protein expressions. Results: C3DM effectively inhibited the proliferation of NSCLC cells. Moreover, C3DM increased the antiproliferative activity of Taxol and significantly enhanced cell apoptosis induced by Taxol in Lewis lung cancer cells. C3DM alone also suppressed Lewis tumor growth and enhanced the antitumor activity of Taxol in vivo. Western blot analysis revealed that the effects of the antiproliferation and apoptosis induction of C3DM treatment alone or in combination with Taxol on Lewis lung cancer were mediated by inhibiting the interleukin-6 (IL-6)/Jak2/STAT3 and IL-6/AKT signaling pathways. Conclusions: The results showed that C3DM has the potential to be used in combination therapy with Taxol against NSCLC.

Keywords: Combination therapy, enhancing antitumor activity, ginsenoside C3DM, nonsmall-cell lung cancer, paclitaxel


How to cite this article:
Tang M, Huang LL, Du QQ, Yan C, Gu AD, Yang JL, Li Y. Ginsenoside 3β-O-Glc-DM (C3DM) enhances the antitumor activity of Taxol on Lewis lung cancer by targeting the interleukin-6/Jak2/STAT3 and interleukin-6/AKT signaling pathways. World J Tradit Chin Med 2020;6:432-40

How to cite this URL:
Tang M, Huang LL, Du QQ, Yan C, Gu AD, Yang JL, Li Y. Ginsenoside 3β-O-Glc-DM (C3DM) enhances the antitumor activity of Taxol on Lewis lung cancer by targeting the interleukin-6/Jak2/STAT3 and interleukin-6/AKT signaling pathways. World J Tradit Chin Med [serial online] 2020 [cited 2021 Jan 25];6:432-40. Available from: https://www.wjtcm.net/text.asp?2020/6/4/432/303582




  Introduction Top


Lung cancer accounts for most cancer fatalities, with >17.6 million deaths worldwide.[1] Approximately 80%–85% of all lung cancers are classified as nonsmall-cell lung cancer (NSCLC). Despite advances in NSCLC treatment, the 5-year relative survival rate of NSCLC is <20%.[2] Paclitaxel (Taxol) is currently a first-line chemotherapeutic drug in clinical oncology and has shown excellent efficacy against advanced NSCLC.[3] However, the potential serious side effects (such as bone marrow suppression and hepatotoxicity) under high therapeutic dosage and chemoresistance have limited the use of Taxol.[4] Therefore, it is an urgent need to explore various combination approaches with low-dose Taxol in the treatment of NSCLC.

Chemotherapy in combination with traditional drugs has attracted considerable attention in cancer therapy, which could effectively promote cytotoxicity of chemotherapeutic drugs, alleviate drug resistance, and enhance the response of patients to chemotherapy.[5],[6],[7] Among the various traditional drugs proposed to improve cancer treatment, ginseng has drawn the most attention worldwide. Ginsenosides have been identified as the main components of ginseng, and protopanaxadiol (PPD)-type ginsenoside showed the greatest anticancer activity among them.[8] Compelling evidence has revealed that ginsenosides combined with chemotherapy (such as Taxol, Temozolomide (TMZ), and cisplatin) effectively inhibited tumor growth and enhanced the sensitivity of chemoresistant tumors to chemotherapeutic drugs.[9],[10],[11]

Although ginsenosides are considered as an excellent option for their anticancer effect in clinical therapy, the sources of ginsenosides are very limited. In recent years, researchers explored an innovative way to produce ginsenosides on a large scale through microbial fermentation, which might accelerate the use of ginsenosides in anticancer drugs.[12],[13] Our previous research has demonstrated that the ginsenoside C3C12PPD produced by microbial fermentation exhibited special anticancer activity in lung cancer.[14] Dammarenediol-II (DM) is the director precursor of PPD. Since DM glucosides have scarcely been identified from Panax species and they are hard to prepare in large quantities, a few studies have investigated the anticancer activity of DM-type ginsenosides in the past few years. Now, researchers have established a green and sustainable approach for the industrial production of DM-type ginsenosides by microbial fermentation, and ginsenoside 3-O-β-D-glucopyranosyl-dammar-24-ene-3β, 20S-diol (3β-O-Glc-DM, C3DM) is one of these DM-type ginsenosides.[15] The anticancer effects and mechanism of the combination of C3DM with chemotherapy drugs in vivo were rarely reported before. This study aims to identify whether C3DM produced by microbial fermentation on a large scale could enhance the effect of Taxol on Lewis lung cancer (LLC) in vitro and in vivo and to further investigate the underlying mechanism of synergistic effects of Taxol with C3DM on Lewis lung cancer.


  Materials and Methods Top


Cell culture

Human lung cancer cell lines NCI-H460, NCI-H1975, NCI-H1650, NCI-H358, and A549 and murine lung cancer cell lines LLC were purchased from the cell center of the Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). Human lung cancer cell lines PC-9 were purchased from Shanghai Fuxiang Biotechnology Co., Ltd. (Shanghai, China). The NCI-H1975, PC-9, A549, and LLC cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the NCI-H1650, NCI-H460, and NCI-H358 cells were cultured in RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc.). All cell cultures were supplemented with 15% fetal bovine serum (FBS; YHSM, Beijing, China), 100 IU/mL penicillin, and 100 μg/mL streptomycin. All cell lines were incubated at 37°C with 5% CO2.

Drugs and compounds

The C3DM was provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (High Performance Liquid Chromatography (HPLC) purity >98%). C3DM was dissolved in dimethyl sulfoxide (DMSO) for in vitro experiments and was dissolved in a solution of 25% PEG400 for in vivo experiments. For the in vivo experiments, Taxol was diluted by 0.9% NaCl prior to be used.

MTT assay

Seven NSCLC cell lines were first incubated in 96-well plates for 24 h (NCI-H460, NCI-H1975, A549, and PC-9 cell lines were incubated with an initial density of 1500 cells per well; NCI-H1650, NCI-H358, and LLC cell lines were incubated with an initial density of 2000 cells per well). Cells were then treated with varying concentrations of C3DM or Taxol. The same fresh medium was added as a vehicle. After incubation for 72–120 h, a total of 50 μL MTT stock solutions (2 mg/mL, BIOFROXX, Germany) were added to each well. The 96-well plates were subsequently incubated for a further 2 h at 37°C. The medium was replaced with DMSO (200 μL/well, Biosharp, Inc., Hefei, China); following gentle agitation, a microplate reader (WD-2102A, Beijing Liuyi Biotech Co., Ltd., China) was used to measure the absorbance at 570 nm. Three parallel samples were measured in each cell line. The absorbance values were normalized to the values obtained for vehicle-treated cells to determine the percentage of surviving cells. The cell inhibition rate (IR) was calculated according to the following formula: IR (%) = (A570 vehicle-treated cells − A570 drug-treated cells)/A570 vehicle-treated cells × 100. The 50% inhibitory concentration (IC50) was assessed using the median effect equation. Dose–response curves were analyzed using Origin 8.5 software (OriginLab, USA). The experiment was repeated three times.

Cell apoptosis assay

LLC cells were treated with 11.1, 33.3, and 100.0 μmol/L C3DM and/or 100.0 nmol/L Taxol for 72 h. Subsequently, cells were harvested using trypsin-ethylenediaminetetraacetic acid (EDTA) solution (0.05% trypsin and 0.02% EDTA in phosphate-buffered saline [PBS]), washed with 1 × PBS twice, and suspended at 2 × 106 cells/mL. Then, the cells were fixed in 70% ethanol at −80°C for >24 h. Treated cells were washed with 1×PBS three times and stained with 500 μl PI staining solution (0.05 mg/mL DAPI, 0.01 mg/mL RNase A, 0.5 mg/mL sodium citrate, and 0.15% Triton X-100, purchased from Sigma, USA). Then, the cells were filtered with 300 mesh screens and incubated for 30 min at room temperature in the dark. A total of 30000 cells of each sample were analyzed using the Accuri C6 flow cytometer (BD, USA).

Lewis lung cancer xenograft mouse model

All animal studies were in compliance with policies of the Institute of Material Medical Animal Care and Use Committee. All animal protocols conformed to the Guidelines for the Care and Use of Laboratory Animals, and the study protocol was approved by the Animal Care and Use Committee of Chinese Academy of Medical Sciences and Peking Union Medical College (Approval no. 0005681).

C57BL/6 mice (males, 15–18 g) were purchased from the National Institutes for Food and Drug Control (Beijing, China). The C57BL/6 mice were subcutaneously implanted with 0.2 mL of the tissue cell suspension (5 × 107 cells/mL) in their left flank. Approximately 24 h after inoculation, the C57BL/6 mice were randomly divided into four groups, with five animals in each group. One group received gavage (p.o.) of 25% PEG400 as a model control; one group received an intraperitoneal injection of 15.0 mg/kg Taxol (once every three days); one group received p.o. of 10.0 mg/kg of C3DM for 12 days; and one group received both 15.0 mg/kg Taxol and 10.0 mg/kg of C3DM for 12 days. When the tumor volume of the control group was about 2000 mm3, the mice were euthanized and tumors were excised, weighed, and photographed. The IR of tumor growth was calculated using the following formula: IR (%) = ([A − B]/A)/100, where A is the average tumor weight of model control and B is the average tumor weight of treatment groups. The tumor tissues were stored at −80°C for subsequent analyses.

Western blot analysis

Tissues were collected from three randomly selected tumors from each group of the Lewis xenograft mice: the model control, 15.0 mg/kg Taxol, 10.0 mg/kg C3DM, and 15.0 mg/kg Taxol combined with 10.0 mg/kg C3DM groups. Tumor tissues were lysed using RIPA buffer (ComWin Biotech Co., Ltd., China) for 40 min on ice, then centrifuged at 12,000 rpm for 15 min at 4°C. Proteins were separated on SDS-PAGE and then transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5% skimmed milk in TBST for 1 h at room temperature and were then incubated overnight at 4°C with the primary antibodies (Bcl-2 antibody, Abcam; the remaining antibodies were purchased from Cell Signaling Technology, Inc., Danvers, MA USA). After washing three times with Tris-buffered saline containing 0.2% Tween®-20 (Beijing Solarbio Science and Technology Co., Ltd.), the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Inc.) and measured using an ECL Western Blot Detection and Analysis System (Applygen Technologies Inc., Beijing, China). β-actin was used as a loading control for membrane proteins. Band intensive sites were analyzed with a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD).

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± standard deviation. Statistical analysis to examine the differences between two groups was performed using an unpaired Student's t-test and analysis to examine the differences among multiple groups was performed using a one-Way ANOVA and Turkey post hoc test. P < 0.05 was considered statistically significant.


  Results Top


C3DM alone or in combination with Taxol inhibited the proliferation of nonsmall-cell lung cancer cells.

The NCI-H460, NCI-H1975, NCI-H1650, NCI-H358, A549, PC-9, and LLC lung cancer cells were treated with 0.032–500.0 μmol/L C3DM for 120 h. The results of the MTT assay showed that the cell viability of seven cell lines was inhibited by C3DM in a dose-dependent manner with IC50 values between 48.01 ± 5.06 μmol/L and 262.85 ± 59.24 μmol/L [Table 1] and [Figure 1].
Table 1: The effect of C3DM on the proliferation of nonsmall-cell lung cancer cells (mean±standard deviation, n=3)

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Figure 1: C3DM inhibited the growth of NSCLC cells analyzed by MTT. Cells were treated with increasing doses of C3DM (0.032-500.0 μmol/L) for 120 h. The results were measured as the mean ± standard deviation and were shown from three independent experiments performed in triplicate

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Three doses of C3DM (11.1, 33.3, and 100.0 μmol/L) were chosen to be used in combination with Taxol (2.54–1851.85 nmol/L) to determine their antiproliferation activity on LLC cells. The results revealed that the IC50 value of Taxol on LLC cells for 72 h was 319.56 ± 49.80 nmol/L, while the IC50 values were reduced to 197.73 ± 38.37 nmol/L, 96.01 ± 8.94 nmol/L, and <2.54 nmol/L, respectively, after incubation with 11.1 μmol/L, 33.3 μmol/L, and 100.0 μmol/L C3DM [Table 2]. The “One-belt, One-line” model was used to evaluate the combination effects according to previous studies:[16] The actual observed dose–effect relationship of the two-drug combination is a black curve in [Figure 2], and the expected additive effect is a value domain with red curves. Synergy is defined as the actual observed efficacy being greater than the value domain range of the expected additive effect, addition is the actual observed efficacy being within the value domain range of the expected additive effect, and antagonism is the actual observed efficacy being less than the value domain range of the expected additive effect. The results showed that when 11.1 μmol/L or 33.3 μmol/L C3DM combined with 2.54 nmol/L or 7.62 nmol/L Taxol, their antiproliferation was antagonistic, while the combination of 11.1 μmol/L C3DM with 68.59 nmol/L Taxol showed additive effects. However, when 11.1 μmol/L or 33.3 μmol/L C3DM was combined with 205.76–1851.85 nmol/L Taxol, they had synergistic effects on each other. Furthermore, the combination of 100.0 μmol/L C3DM with variable doses of Taxol in the experimental range had synergistic inhibition on the proliferation of LLC cells [Table 3] and [Figure 2].
Table 2: The effect of Taxol combined with C3DM on the proliferation of Lewis lung cancer cells (mean±standard deviation, n=3)

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Table 3: Combination index based on dose (CId) of ginsenoside C3DM combined with taxol

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Figure 2: Dose–effect belt of the expected addictive effects and the actual observed dose–effect curve for combination of C3DM and Taxol on Lewis lung cancer cells. (a) 11.1 μmol/L of C3DM combined with Taxol. (b) 33.3 μmol/L of C3DM combined with Taxol. (c) 100.0 μmol/L of C3DM combined with Taxol

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C3DM enhanced the apoptosis induced by Taxol on LLC cells

In view of the synergistic effect of C3DM and Taxol on the proliferation, we then determined the effect of them on apoptosis of LLC cells by flow cytometry. The results showed that individual Taxol treatment (100.0 nmol/L) had a little effect on apoptosis (the apoptosis rate was 19.37% ± 2.89%), and individual C3DM (11.1, 33.3, and 100.0 μmol/L) treatment induced apoptosis of LLC cells <10% (5.67% ± 0.93%, 7.03% ± 3.44%, and 7.33% ± 1.66%, respectively). However, combined treatment with 11.1, 33.3, and 100.0 μmol/L C3DM and Taxol showed the synergistic effect on inducing apoptosis, and the apoptosis rate increased to 24.23% ± 4.19% (P < 0.001), 31.87% ± 2.34% (P < 0.001), and 66.37% ± 6.25% (P < 0.001), respectively [Figure 3].
Figure 3: The synergistic effect of C3DM and Taxol on apoptosis of Lewis lung cancer cells analyzed by FCM. (a) The apoptosis of Lewis lung cancer cells incubated with Taxol and C3DM for 72 h. (b) Columns indicated the apoptosis rate of Lewis lung cancer cells. The results were measured as the mean ± standard deviation. Each sample was repeated in three times. ***P < 0.001 versus DMSO; ##P < 0.01, ###P ? 0.001 versus Taxol; △△△P < 0.001 versus 11.1 μmol/L C3DM; ▽▽▽P < 0.001 versus 33.3 μmol/L C3DM; ◊◊◊P < 0.001 versus 100.0 μmol/L C3DM

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C3DM promoted antitumor effect of Taxol on Lewis lung cancer xenograft model

In order to further explore the combination therapy in vivo, we chose the dose of 15.0 mg/kg Taxol with about 40% ~50% IR against Lewis lung cancer to combine with C3DM to study the growth inhibition effect on the mouse transplanted tumor model. As in our previous study, 10.0 mg/kg C3DM was chosen for this study too. Tumor growth was suppressed in mice upon administration with 15.0 mg/kg Taxol or 10.0 mg/kg C3DM alone, with the tumor IR of 40.1% and 44.6% (P < 0.05), respectively, according to the tumor weight. Interestingly, the combination of two compounds significantly inhibited the tumor growth with the IR of 80.8% (P < 0.001), suggesting that C3DM could further enhance the antitumor activity of Taxol on Lewis lung cancer xenograft model [Table 4] and [Figure 4].
Table 4: C3DM promoted antitumor effect of taxol on Lewis lung cancer xenograft model

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Figure 4: C3DM promoted anti-tumor effect of Taxol on Lewis lung cancer xenograft model. (a) Representative image of the tumor. (b and c) Tumor weight. The results were measured as the mean ± standard deviation. *P < 0.05, ***P < 0.001 versus control

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C3DM combined with Taxol influenced interleukin-6/Jak2/STAT3 and interleukin-6/AKT signaling pathways activity

Interleukin-6 (IL-6) influences the Jak2/STAT3 and PI3K/AKT signaling pathways, which are known to be involved in the regulation of multiple cellular functions (such as growth, differentiation, and proliferation survival) and play a key role in tumorigenesis and chemoresistance.[17],[18],[19] Therefore, the present study detected the expressions of the protein associated with these pathways in Lewis tumors after treatment with 15.0 mg/kg Taxol and/or 10.0 mg/kg C3DM.

At first, we examined the IL-6/Jak2/STAT3 pathway. The results of western blotting showed that Taxol treatment alone had no effect on this pathway, while C3DM inhibited IL-6 protein and its downstream proteins of p-Jak2, Jak2, p-STAT3 (Tyr705), and STAT3 were also markedly reduced. Meanwhile, the combination group further suppressed the protein expressions of IL-6, p-Jak2, Jak2, p-STAT3 (Tyr705), and STAT3 [Figure 5]a. Next, we examined the AKT pathway. The results displayed that Taxol had no significant effect on the phosphorylation of AKT (Thr 308) under the dose of 15.0 mg/kg, while both C3DM and the combination group of Taxol with C3DM observably suppressed the phosphorylation of AKT (Thr 308) and total protein of AKT [Figure 5]b.
Figure 5: The effect of C3DM on the related protein expression in Lewis cells xenograft tumors analyzed by western blotting. (a) IL-6/Jak2/STAT3 signaling pathway; (b) AKT signaling pathway; (c) The proteins related to cell proliferation and apoptosis. (d) The ratio of Bcl-2/Bax of each group. The results were measured as the mean ± standard deviation. Each sample was repeated in three times. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Taxol; △P < 0.05 versus C3DM

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c-Myc is a key regulator of cell proliferation, cell growth, differentiation, and apoptosis.[20] As showed in [Figure 5]c, the c-Myc protein level of the combination therapy group was significantly lower than that observed when treated with Taxol or C3DM alone. The Bcl-2 and IAP protein families play pivotal roles in cell apoptosis by influencing a wide array of diverse upstream survival and distress signals to decide cell fate.[21] As indicated in [Figure 5]c, the combination therapy group showed significant downregulation of the Bcl-2, c-IAP1, and c-IAP2 activities compared with Taxol or C3DM treatment alone, while the protein level of Bax was further increased following the administration of C3DM and Taxol together. Meanwhile, the ratio of Bcl-2/Bax was markedly decreased in the C3DM group and the combination therapy group [Figure 5]d.


  Discussion Top


Combination therapy for cancer is aimed at obtaining a better outcome and is commonly used in the clinical setting.[22] Taxol is a microtubule-stabilizing therapeutic drug that is approved for the treatment of ovarian, breast, and lung cancer.[23] Combining Taxol with other traditional agents is a promising strategy to increase the chemosensitivity of Taxol. Ginsenoside is a popular herbal medicine used to promote body immunity, vitality, and longevity. Anticancer activity of ginsenoside Rg3 has been broadly researched for the past few years, and it is clinically approved for use and prescribed as an adjuvant treatment in multiple cancer therapies in China.[24] A previous study revealed that the combination of Rg3 with docetaxel inhibited the susceptibility of prostate cancer cells,[25] and Rg3 promoted cytotoxicity of paclitaxel (Taxol) on triple-negative breast cancer by regulating Bax/Bcl-2 expression.[6] The anticancer effects of C3DM, a ginsenoside with a similar chemical structure to Rg3, was rarely investigated, partly due to the insufficient production of the compound. Now, C3DM can be prepared on a large scale through metabolic engineering, as shown by Professor Yang,[15] which makes it possible for us to conduct research related to efficacy evaluation and mechanism of C3DM. In this study, we focused on exploring how C3DM enhanced the anticancer effects of Taxol on Lewis lung cancer.

At first, combination studies were designed and executed to assess interaction (synergistic, additive, or antagonistic effects) between the C3DM and Taxol in vitro according to the method of “One-belt, One-line.”[16] Different doses of C3DM (11.1, 33.3, and 100.0 μmol/L) in combination with variable doses of Taxol showed an antagonistic, additive, or synergistic effect on LLC cells. We confirmed that 68.59–1851.85 nmol/L Taxol in combination with C3DM exerted additive or synergistic effects on LLC cells. Thus, we chose 100.0 nmol/L Taxol combined with C3DM to detect the effect on apoptosis, and the results proved that C3DM also promoted the apoptotic effect of Taxol on LLC cells. These data from in vitro experiments laid a solid experimental foundation for us to further explore the combination therapy in vivo. We used the Lewis lung cancer xenograft model for in vivo experiments with low-dose Taxol (40%~50% IR) and the suitable dosage of C3DM according to results from our previous study. We proved that 15.0 mg/kg Taxol combined with 10.0 mg/kg C3DM had a better inhibitory activity (80.8%) than Taxol (40.1%) or C3DM alone (44.6%).

In consideration of the efficacy of C3DM on promoting the antitumor activity of Taxol in vitro and in vivo, we further investigated the mechanism. IL-6, a pleiotropic cytokine, mediates its downstream effects by activating a number of signaling cascades including JAK/STAT, PI3K/AKT, and MAPK pathways.[26],[27] IL-6/JAK2/STAT3 signaling pathway has long been associated with tumorigenesis, and persistent phosphorylation of STAT3 has been observed in 22%~65% of NSCLC.[28] Moreover, STAT3 has been proven to be an important therapeutic target in cancer,[29] and previous studies have indicated that agents targeting STAT3 showed promising signals of efficacy in the treatment of NSCLC, hepatocellular carcinoma, and multiple myeloma.[30],[31],[32] Ginsenoside 20(S)-Rh2 was reported to exhibit effective cytotoxic activity by inhibiting the IL-6-induced JAK2/STAT3 pathway in human colorectal cancer cells,[33] which is consistent with our findings that C3DM significantly inhibited IL-6, p-Jak2, p-STAT3 (Tyr705), and STAT3 in Lewis tumor. AKT signaling pathway is also involved in NSCLC development.[34] It can be deduced that ginsenoside Rg3 inhibited cell viability and induced apoptosis through the PI3K/AKT signaling pathway in lung cancer cells.[35] Similarly, our previous work demonstrated that a biosynthetic ginsenoside C3C12PPD could effectively inhibit the proliferation and migration of lung cancer cells through Raf/MEK/ERK, AKT/mTOR, and AKT/GSK-3β/β-Catenin signaling pathways.[14] Now, we found that C3DM also could significantly inhibit phosphorylation of AKT. In addition, the proto-oncogene c-Myc, Bcl-2, and IAP protein families correlated with cell proliferation and apoptosis are at the downstream of IL-6/JAK2/STAT3 and AKT signaling pathways;[36],[37] our present results indicate that C3DM downregulates IL-6/JAK2/STAT3 and AKT pathways accompanied with the inhibition of c-Myc, Bcl-2, and c-IAP1/2, and promoting the level of Bax in Lewis tumor, which could partly account for the antiproliferation and induced apoptosis effects of C3DM on NSCLC.

There is evidence that upregulation of IL-6 promotes multidrug resistance through activation of JAK/STAT3 and PI3K/AKT pathways.[38] Several earlier studies suggested that IL-6/Jak2/STAT3 signaling pathway plays a vital role in Taxol resistance of cancer cells, and inhibiting this pathway could increase Taxol sensitivity.[39],[40] Hence, we hypothesized that C3DM might increase the activity of Taxol on lung cancer through IL-6/Jak2/STAT3 and IL-6/AKT pathways also. Our study showed that C3DM in combination with Taxol further inhibited the expression of IL-6, p-AKT, p-Jak2, and p-STAT3, STAT3 in Lewis tumor tissues, suggesting that C3DM increased the chemosensitivity to Taxol and this was associated with IL-6/Jak2/STAT3 and IL-6/AKT signaling pathways. Activation of STAT3 and AKT has been known to induce resistance to apoptosis in NSCLC.[41],[42] We observed that the combinational treatment significantly potentiated Taxol-induced apoptosis. Furthermore, we observed downregulation of c-Myc, Bcl-2, and c-IAP1/2 and upregulation of the ratio of Bcl-2/Bax, which was regulated by STAT3 and AKT. A previous study showed that Taxol treatment alone did not affect p-Jak2 and p-STAT3 (Tyr705) in A549 tumor tissues, while Taxol combination with a STAT3 inhibitor could suppress the phosphorylation of Jak2 and STAT3, and all the treatment groups had no effects on total Jak2 and STAT3.[30] In the present study, we found that Taxol treatment did not influence the expression of p-Jak2, but the level of p-STAT3 (Tyr705), total Jak2, and STAT3 was higher in the Taxol group than the control group in Lewis tumor tissues, which was different from the results of the previous study. Therefore, the relation of the JAK2/STAT pathway with the chemoresistance to Taxol needs to be more closely investigated in future research.

Taken together, these findings indicated that C3DM combined with Taxol exerted a synergistic effect on inhibiting proliferation and inducing apoptosis of NSCLC, which might be associated with IL-6/Jak2/STAT3 and IL-6/AKT signaling pathways.


  Conclusions Top


In the present study, we proved that C3DM combined with Taxol showed antiproliferative and apoptotic effect and enhanced the antitumor activity of Taxol on Lewis lung cancer in vitro and in the mouse model by inhibiting IL-6/Jak2/STAT3 and IL-6/AKT signaling pathways, which made an experimental basis for its development as a novel adjuvant agent.

Financial support and sponsorship

The study was supported by the Chinese Academy of Medical Sciences (CAMS) Initiation fund for Medical Science (2016-I2M-1-008) and the National Natural Science Foundation of China (Approval no. 81673341).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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