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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 2  |  Page : 324-330

Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice


Department of Respiratory Medicine, The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China

Date of Web Publication7-Jul-2015

Correspondence Address:
Gang Chen
Department of Respiratory Medicine, The Third Hospital of Hebei Medical University, No. 139 Ziqiang Road Qiaoxi, Shijiazhuang - 050051, Hebei
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.151444

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 > Abstract 

Objective: This work was aimed at studying the inhibitory activity of metformin combined with the commonly used chemotherapy drug cisplatin in human lung cancer xenografts in nude mice. We also examined the combined effects of these drugs on the molecular expression of survivin, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factorreceptor-3 (VEGFR-3) to determine the mechanism of action and to explore the potential applications of the new effective drug therapy in lung cancer.
Materials and Methods: The nude mice model of lung cancer xenografts was established, and mice were randomly divided into the metformin group, the cisplatin group, the metformin + cisplatin group, and the control group. The animals were killed 42 days after drug administration, and the tumor tissues were then sampled to detect the messenger ribonucleic acid (mRNA) and protein expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).
Results: The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the cisplatin group and the combined treatment group were lower than that in the control group (P < 0.05). In the metformin group, the expression of MMP-2 protein and mRNA was lower than that in the control group (P < 0.05). The protein and mRNA expression levels of survivin, MMP-2, VEGF-C, and VEGFR-3 in the combined treatment group were lower than that in the cisplatin group and the metformin group (P < 0.05).
Conclusions: Metformin inhibited the expression of MMP-2, cisplatin and the combined treatment inhibited the expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the combined treatment of metformin with cisplatin resulted in enhanced anti-tumor efficacy.

Keywords: Cisplatin, lung cancer, matrix metalloproteinase-2, metformin, survivin, vascular endothelial growth factor -C, vascular endothelial growth factor receptor-3


How to cite this article:
Chen YQ, Chen G. Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice. J Can Res Ther 2015;11:324-30

How to cite this URL:
Chen YQ, Chen G. Combined therapeutic effect and molecular mechanisms of metformin and cisplatin in human lung cancer xenografts in nude mice. J Can Res Ther [serial online] 2015 [cited 2019 Nov 15];11:324-30. Available from: http://www.cancerjournal.net/text.asp?2015/11/2/324/151444


 > Introduction Top


Recent studies have found that metformin could reduce the risk of malignant tumors in diabetic patients, inhibit tumor cell growth, and enhance the effects of chemotherapy drugs. Its anti-tumor effect and potential use as an anti-cancer drug has attracted attention in the fields of diabetes and cancer research. Recent clinical studies have found that the incidence of tumors in diabetic patients who took metformin is significantly decreased. [1] Hence, the application of metformin has been of interest in cancer research and has gradually become a highlight of these studies. Certain studies have shown that the combination of metformin and cisplatin could have a synergistic effect, which would be helpful to inhibit resistance to cisplatin. [2] However, the mechanism underlying this effect still requires further research. In this study, we explored the combined effects of metformin and cisplatin in human lung cancer xenografts in nude mice and examined their effect on the expression of survivin, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factor receptor-3 (VEGFR-3) in tumors to determine the molecular mechanisms that may be useful for lung cancer treatment.


 > Materials and Methods Top


Cell cultivation

The human lung adenocarcinoma cell line A549 was used for cell cultivation in F12k medium, which contained 12% imported fetal bovine serum, 100U/ml penicillin, and 100U/ml streptomycin. The incubation was performed at 37°C and 5% CO 2 .

Cell seeding

Thirty-three male BALB/c nude mice, weighing 20-24 g and aged 4-5 weeks, were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China); the certificate number for experimental animals was SCXK Jing-204-0004. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Hebei Medical University. The animals were fed sterile feed and sterile purified water, and kept in individually ventilated cages, with the appropriate ambient temperature and humidity.

A single cell suspension of the logarithmically growing A549 cells was prepared, and 0.2 ml of the suspension was inoculated subcutaneously into the left axilla of disinfected nude skin using a 1-ml syringe.

Grouping and treatment

When the average diameter of xenografts was approximately 4 mm, the mouse with the smallest tumor was removed, and the remaining nude mice were randomly divided into the control group, the metformin group, the cisplatin group, and the metformin + cisplatin group, with eight mice per group. The cisplatin group was intraperitoneally administered cisplatin injection (Nanjing Pharmaceutical Co., Nanjing, China, Approval Number: Zhunzi H20030675), at a dose of 5 mg/kg once per week at the same time. The mice were also orally administered sterile distilled water. The metformin group was orally administered metformin (Sino-American Shanghai Squibb Pharmaceuticals Ltd., Shanghai, China, approval number: Zhunzi H20023371), at a dose of 200 mg/kg/d, at the same time each day, and were intraperitoneally injected the same volume of saline. The combination group was orally administered metformin (200 mg/kg/d) and intraperitoneally injected cisplatin (5 mg/kg once per week). The control group was orally administered the same amount of sterile distilled water and intraperitoneally injected the same amount of saline. Forty-two days later, the mice were killed using the cervical dislocation method for tumor tissue sampling. The samples were fixed in 4% paraformaldehyde, and sections were conventionally embedded for immunohistochemical detection. Some tissue samples were placed in Eppendorf tubes containing ribonucleic acid (RNA) preservation solution and frozen with liquid nitrogen for further polymerase chain reaction (PCR) studies.

Immunohistochemistry

The sampled tumor tissues were fixed in a 4% paraformaldehyde solution, and subjected to a gradient alcohol dehydration (the alcohol concentrations were 75%, 80%, 90%, 95% ×2, 100% ×2, xylene × 2, applied in a gradient). The sections were then dipped into wax twice for the paraffin embedding.

The self-potential (SP) method was used. The slices were 4-μm thick, and conventional dewaxing was performed; when the temperature of the antigen repairing liquid was higher than 93°C, the slices were immersed in 0.01 mol/L citrate buffer for 12 min for repair, followed by a 45-min cooling-down at room temperature. The slices were then soaked thrice in distilled water for 5 min each, followed by incubation in 3% H 2 O 2 at 37°C for 20 min to remove endogenous peroxidase activity. The slices were then soaked once in distilled water for 5 min and thrice in Phosphate buffered saline (PBS) for 5 min each. The primary antibody was incubated overnight at 4°C. Survivin rabbit anti-human polyclonal antibody (the concentrations of antibodies were all 1:200) and MMP-2 rabbit anti-human polyclonal antibody (1:200) were purchased from Bioworld Co. (Nanjing, China), VEGF-Crabbit anti-human monoclonal antibody (1:200) was purchased from Boster Co. (Wuhan, China), and VEGFR-3 mouse anti-human monoclonal antibody (1:200) was purchased from Zhongshan Co. (Beijing, China). The slices were then immersed thrice in PBS for 5 min each, and were incubated with the second antibody at 37°C for 30 min. 3,3'-Diaminobenzidine (DAB) staining was then performed; it was terminated when the specific region exhibited the maximum expression under a microscope. The slices were subsequently rinsed thrice in water for 10 min each, restained with hematoxylin for 1 min, restained with blue dye for differentiation for 30 s, and dehydrated and mounted. PBS, instead of the primary antibody, was used as the negative control for sample staining. The positive biopsy provided by the company was used as the positive control, and the samples were observed and photographed under an optical microscope. The second antibody and DAB were purchased from Zhongshan Co. (Beijing, China). The survivin-positive products were located in the cytoplasm or nucleus, and a brownish-yellow color indicated a positive result. The MMP-2-positive products were located in the membrane and cytoplasm, and a brownish-yellow color indicated a positive result. The VEGF-C- and VEGFR-3-positive products were located in the cytoplasm, and brownish-yellow colors indicated positive results. The tissue sections were first observed under a low magnification, followed by light microscopy at 200× magnification. Ten images were randomly observed, and the Computer color magic analysis system (CMIAS) true color pathological image analysis system was used to calculate the average integrated optical density. For the imaging analysis, the instrument was composed of an image scanner, image tube, and image processer (computer). The imaging analysis instrument had its own photodensitometer, which could transform the visual signals obtained in the image scanner into log values. The so-called integrated optical density was the sum of singly detected pixel density distributions, and by measuring the area of the target subject, the average optical density was calculated.

Reverse transcription polymerase chain reaction (RT-PCR)

The primers were synthesized by Shanghai Biological Engineering Co., Ltd. (Shanghai, China). The primer sequences were as follows: GAPDH upstream primer, 5'-TGAACGGGAAGCTCACTGG-3'; downstream primer, 5'-GCTTCACCACCTTCTTGATGTC-3'; survivin upstream primer, 5'-TTTCTCAAGG-ACCACCGCA-3'; downstream primer, 5'-AGTCTGGCTCGTTCTCAGTG-3'; MMP-2 upstream primer, 5'-AAC-TACGATGATGACCGCAAG-3'; downstream primer, 5'-GACAGACG-GAA-GTTCTTGGTG-3'; VEGF-C upstream primer, 5'-CAATCACACTTCCTGCCG-ATG-3'; downstream primer, 5'-GGTCTTGTTCGCTGCCTGA-3'; VEGFR-3 upstream primer, 5'-GCAGACCCACACAGAACTCT-3'; downstream primer, 5'-TCGCTGGATGCCGTTGTT-3'.

For the extraction of total tissue RNA, 50 mg tissue was used according to the following procedure: 1 ml Trizol and 50 mg tissue were added to an ice-bath homogenizer for rapid grinding, and the mixture was kept still at room temperature for 5 min. Chloroform (200 ml) was then added and the mixture was shaken vigorously for 15 s at 4°C and 12,000 rpm for 15 min. The upper aqueous phase was transferred to another Eppendorf tube and an equal volume of isopropanol was added and slightly mixed for 15 s. The mixture was then centrifuged at 4°C and 12,000 rpm for 10 min; the supernatant was then discarded, and 1,000 ml of 75% ethanol was added to the tube and mixed gently at 4°C, 7,500 rpm for 5 min. The mixture was dried; 30 ml Diethy pyrocarbonate (DEPC)-treated deionized water was then added to dissolve the precipitate, which was stored at -80°C for future detection.

Quantitation of the extracted RNA: The 756 ultraviolet (UV) spectrophotometer was used to detect the OD 260 /OD 280 ratio, and to determine the purity and amount of RNA. The sample with an OD 260 /OD 280 ratio of 1.8-2.0 was used for RNA reverse transcription. The RNA concentration was detected by a UV spectrophotometer.

To synthesize the complementary deoxyribonucleic acid (cDNA) first chain, an Eppendorf gene amplification instrument (Hamburg, Germany) was used. The composition of the cDNA first-chain synthesis solution was as follows: 5 × RT buffer 5 μl, concentration 1×; dNTP 2.5 μl, concentration 10 mM; RNasin 2 μl, concentration 20U/l; RNA 5.3 μl, concentration 8 μg; randomprimer 1 μl, concentration 0.1 g/l; M-MLV 6 μl, concentration 5 U/l; nuclease-free water 3.2 μl, with a total volume of 25 μl. The 5 × RT buffer contains the following: 250 mM Tris-HCl (pH 8.3), 250 mM KCl, 50 mM MgCl 2 , 50 mM DTT, and 2.5 mM spermidine.

Reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase was inactivated at 95°C for 5 min.

The RT products consisted of 1 μl, 2 × Master Mi × 10 μl, 1 μl upstream and downstream primer, H 2 O 2 7 μl, with a total volume of 20 μl. Syber Green fluorescence quantitative PCR detection was performed using Hot Start Fluorescent PCR Core Reagent Kits (SYBR Green I, BBI). The thermal cycling parameters were as follows: 95°C for 10 min, followed by the subsequent three steps: 95°C for 15 s, 58°C for 20 s, 72°C for 27 s for 40 cycles. The fluorescence signal of each cycle was collected at the third step (72°C for 27 s).

After the amplification, the results were analyzed in the analysis interface; GAPDH was used as the internal reference gene, and compared with the control group, andthe relative quantification value (RQ value) of the target gene expression was for statistical analysis. The RQ value within the RT-PCR detection was auto-calculated by the PE ABI 7300 Real-Time PCR System (Foster City, CA, USA). The RQ value was the C T value of the comparison, which was derived from the deformation of the mathematical equation during the PCR reaction. The C T value referred to the repeated cycle number when the fluorescence signal in each reaction tube reached a set threshold, and it was used to judge the changes in the expression of the target sites relative to the same sites within the standard samples. The concrete calculation method was as follows: Fold change = 2-ΔΔC T ; ΔΔC T = (C T target gene- C T internal standard) treatment group-(C T target gene- C T internal standard) non-treatment group.

Statistical analysis

The statistical analysis was performed using the statistical package of social sciences (SPSS) 13.0 software. The data were expressed as means ± standard deviation (SD), and analysis of variance (ANOVA) was used to compare group means; P < 0.05 considered statistically significant.


 > Results Top


0Expression of survivin, MMP-2, VEGF-C, and VEGFR-3 proteins

Positive survivin expression inside tumor cells was mainly found as brown particles in the nucleus or cytoplasm, [Figure 1]a-d, [Table 1]. The survivin protein levels of the cisplatin group and the metformin + cisplatin combination group were lower than the control group (P < 0.05), while that of the metformin group did not differ from the control group (P > 0.05). The survivin protein level for the combination treatment group was lower than it was for the metformin group or the cisplatin group (P < 0.05).
Figure 1: Expression of survivin protein, MMP-2 protein, VEGF-C protein, and VEGFR-3 protein in control, metformin-treated, cisplatin-treated, and metformin combined with cisplatin-treated group in xenografts tumor of lung cancer (A549) in nude mice (IHC, ×200). (a-d) Expression of survivin protein, e-h) Expression of MMP-2 protein, (i-l) Expression of VEGF-C protein, (m-p) Expression of VEGFR-3 protein. MMP-2 = Matrix metalloproteinase-2, VEGF-C = Vascular endothelial growth factor-C, VEGFR-3 = Vascular endothelial growth factorreceptor-3

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Table 1: The expression of survivin, MMP - 2, VEGF - C, VEGFR - 3 protein value in different groups of lung cancer (A549) xenografts in nude mice (x¯±s) (The values were the average integrated optical densities, and the units were arbitrary units)

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Positive MMP-2 expression in tumor cells was mainly found as brown particles in the membrane or cytoplasm [Figure 1]e-h, [Table 1]. The MMP-2 protein levels of the metformin group, the cisplatin group, and the combined treatment group were lower than that of the control group (P < 0.05). The MMP-2 protein expression levels of the metformin group and the cisplatin group did not differ from the control group (P > 0.05). The MMP-2 protein level of the combined treatment group was lower than that of the metformin or cisplatin group (P < 0.05).

The positive VEGF-C expression in tumor cells was located in the cytoplasm, with brown granules determined as positive staining [Figure 1]i-l, [Table 1]. The VEGF-C protein levels of the cisplatin group and the combined treatment group were lower than in the control group (P < 0.05), while that of the metformin group did not differ from the control group (P > 0.05). Furthermore, the VEGF-C protein level of the combined treatment group was lower than that of the metformin or cisplatin group (P < 0.05).

The positive VEGFR-3 expression in tumor cells was located in the cytoplasm, with brown granules determined as positive [Figure 1]m-p, [Table 1]. The VEGFR-3 protein levels of the cisplatin group and the combined treatment group were lower than the control group (P < 0.05), while that of the metformin group did not differ from the control group (P > 0.05). The VEGFR-3 protein expression level of the combined treatment group was lower than that of the metformin or cisplatin group (P < 0.05).

Expression of survivin, MMP-2, VEGF-C, and VEGFR-3 mRNA

The survivin mRNA levels of the cisplatin group and the metformin + cisplatin combination group were lower than the control group [P < 0.05, [Table 2]], while that of the metformin group did not differ from the control group [P > 0.05, [Table 2]]. The survivin mRNA level of the combined treatment group was lower than that of the metformin or cisplatin group [P < 0.05, [Table 2]].
Table 2: The expression of survivin, MMP - 2, VEGF - C, VEGFR - 3 mRNA value in different groups of lung cancer (A549) xenografts in nude mice (x¯±s)

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The MMP-2 mRNA levels of the metformin group, the cisplatin group, and the metformin + cisplatin combination group were lower than the control group [P < 0.05, [Table 2]]. The MMP-2 mRNA level of the combined treatment group was lower than that of the metformin or cisplatin group [P < 0.05, [Table 2]].

The VEGF-C mRNA levels of the cisplatin group and the metformin + cisplatin combined treatment group were lower than the control group [P < 0.05, [Table 2]], while that of the metformin group did not differ from the control group [P > 0.05, [Table 2]]. The VEGF-C mRNA level of the combined treatment group was lower than that of the metformin or cisplatin group [P < 0.05, [Table 2]].

The VEGFR-3 mRNA levels of the cisplatin group and the metformin + cisplatin combination group were lower than the control group [P < 0.05, [Table 2]], while that of the metformin group did not differ from the control [P > 0.05, [Table 2]]. The VEGFR-3 mRNA level of the combined treatment group was lower than that of the metformin or cisplatin group [P < 0.05, [Table 2]].


 > Discussion Top


Lung cancer is a leading cause of death worldwide, and approximately one million people, including 850,000 men and 330,000 women, die of lung cancer every year. More than 60% of non-small cell lung cancer patients are in the late stage or exist the metastasis when they are diagnosed, and have no chance of operation. [3] Therefore, new strategies for the treatment of lung cancer are necessary to delay its progress and improve survival rate, and thus improve the patients' quality of life.

Metformin is an oral hypoglycemic drug in the class of biguanides, and is widely used to treat type 2 diabetic patients with body mass index (BMI) >25 kg/m 2 and patients with insulin resistance. Recent studies have also found that metformin could reduce the risk of malignant tumors in diabetic patients, inhibit tumor cell growth, and enhance the effects of chemotherapy drugs. Its application as an anticancer drug has attracted attention in diabetes and cancer research.

Several recent studies have shown that metformin might have a role in reducing the incidence of cancer by various mechanisms: 1) It might directly inhibit tumors via the ATM-LKB1-AMPK-mTOR pathway. [4] The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinase and belongs to an important class of signal transduction pathway molecules that mediate gene expression and cell proliferation, mainly regulating cell growth by controlling protein synthesis. It is also closely associated with tumor angiogenesis. Some studies have shown that low mTOR expression results in reduced expression of various downstream molecules, including VEGFA and cyclinD1. Therefore, reducing mTOR expression could inhibit tumor growth. Metformin could inhibit the expression of mTOR, thus causing tumor inhibition. 2) Metformin could reduce insulin levels; this indirect mechanism might be an important for inhibiting malignant tumors. 3) Metformin could induce cell cycle arrest and apoptosis, and reduce growth factor signaling. [5] 4) Metformin could reduce protein levels inside the cell cycle. 5) It might inhibit the proto-oncogene HER2. [6] 6) It may exert anti-inflammatory effects. 7) It may improve the memory of T cells. [7] In addition to these, there may be other potential mechanisms.

The results of this research showed that cisplatin inhibited survivin, MMP-2, VEGF-C, and VEGFR-3 expression, metformin inhibited MMP-2 expression, and metformin had a synergistic effect with cisplatin on tumor inhibition.

Survivin is a recently discovered unique bifunctional protein that belongs to the inhibitor of apoptosis protein family, and can inhibit programmed cell death (apoptosis) and regulate cell division. [8] It is expressed in the G2/M phase of the cell cycle [9] and regulates angiogenesis, immune signaling, and tumor metastasis. [10] The overexpression of survivin can be carcinogenic, because it affects the regulation of the G2/M phase, thus increasing mitosis and the number of cells. The inhibition of survivin would then prevent its anti-apoptotic effect and mitotic progression in non-small cell lung cancers. [9] This study indicated that the metformin alone did not cause significant inhibition of survivin expression, but when combined with the chemotherapeutic drug cisplatin, it reduced the level of survivin, which effects the cell cycle; thus, survivin has been shown to have anti-tumor and anti-mitosis effects. The specific mechanism of the synergic effects of metformin and the chemotherapeutic drug is still unclear, but a recent study indicated that metformin effects tumor stem cells and the mTOR signaling pathway, thus increasing the sensitization towards chemotherapeutic drugs. [11] It could also influence the cell cycle, thus increasing the sensitivity of tumor cells to chemotherapeutic drugs. [12]

Distant metastasis is the major cause of cancer death. Degradation of the extracellular matrix is necessary for the formation of metastases, resulting in the invasion and infiltration of tumors into local tissues, followed by effusion outside the vessels and the formation of new metastases. [13] Some studies have demonstrated the important roles of enzymes during metastasis, such as the MMPs, which degrade the components of the extracellular matrix and basement membrane and thus facilitate the separation of tumor cells, penetration through tissue barriers, and invasion into adjacent tissues. [14] MMPs are involved in all stages of tumor development, i. e., not only tumor invasion and metastasis but also proliferation, adhesion, migration, differentiation, angiogenesis, aging, cellular autophagy, apoptosis, and immune system escape. [15] The blockage of tumor cell MMP expression could significantly reduce tumor invasion and metastasis. [16] This study indicated that metformin inhibits the expression of MMP-2, thus inhibiting the growth and metastasis of tumors, and playing an anti-tumor role. Currently, there are only few studies on the impacts of metformin on MMP-2 expression, but one study showed that metformin inhibits the invasion of melanomas and inhibits the expression of MMP-2. [17] By inhibiting the expression of MMP-2, metformin plays an anti-cancer role. [18]

VEGF-C and VEGFR-3 play important roles in the formation of tumor blood vessels and lymphatic vessels. VEGF-C consists of 419 amino acid residues, and after proteolytic processing, VEGF-C forms a disulfide-linked homodimer that binds the corresponding receptor and has biological functions. In normal tissues, VEGF-C is a specific lymphangiogenesis-promoting factor, VEGFR-2 is expressed in vascular endothelial cells and the lymphocytic endothelium, and VEGFR-3 is expressed only in the lymphocytic endothelium. This study showed that metformin alone does not significantly inhibit VEGF-C and VEGFR-3 expression, but has a synergic effect with cisplatin; the combined treatment reduced the expression of VEGF-C and VEGFR-3, which has been shown to inhibit neovascularization. [19] Furthermore, metformin inhibits tumor angiogenesis by blocking the mTOR signaling pathway. [20] One study showed that metformin helps to overcome tumor drug resistance by targeting STAT3. Metformin suppresses STAT3 activation via LKB1-AMPK-mTOR-independent but reactive oxygen species (ROS)-related and autocrine interleukin (IL)-6 production-related pathways. [2]

The synergic effects of metformin with cisplatin might occur by a number of mechanisms. Activation of the mTOR signal transduction pathway could inhibit apoptosis induced by various stimuli and promote cell cycle progression, thus promoting cell survival and proliferation. Alternatively, it could be involved in angiogenesis, thus playing an important role in tumor formation, as well as tumor invasion and metastasis. Therefore, it has become a new target for cancer intervention. Some studies have shown that the specific-mTOR inhibitor could effectively improve the sensitivity of chemotherapeutic drugs such as cisplatin, gemcitabine, [21] 5-fluorouracil, and mitomycin, which inhibit the mTOR signaling pathway, enhance the chemotherapeutic effects of cholangiocarcinoma cells, and improve chemotherapy-induced apoptosis, resulting in a synergistic effect. [22] Targeted therapy by inhibiting the PI3K pathway (mTOR) has been demonstrated to have a clinical benefit for the treatment of breast cancer. [23] Metformin could inhibit mTOR, thus playing a synergistic role with chemotherapeutic drugs, but more evidence and in-depth studies are necessary to reveal its benefits.

The immunohistochemistry and real-time PCR results showed that metformin inhibits the expression of MMP-2, suggesting that the anti-tumor effect of metformin might be associated with the regulation of the MMP-2 gene. The combined treatment group had lower expression of survivin, MMP-2, VEGF-C, and VEGFR-3, and the inhibitory effects observed in the combined treatment group were much more obvious than those of the mono-drug group. Therefore, the combination of these two drugs could reduce the toxic side effects of cisplatin and enhance the anti-tumor effect.

 
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