α-Solanine Sensitizes Lung Adenocarcinoma Cells To Cisplatin And X-ray

Background and objectiveLung cancer is the leading cause of cancer associated death worldwide. As an important subtype of lung cancer, lung adenocarcinoma threatens human health with an increasing incidence. Usually the radiosensitivity and chemosensitivity of lung adenocarcinoma turn out to be terribly poor. Although recently some novel strategies have been developed concerning cancer therapy, unluckily the survival rate of lung adenocarcinoma almost remains unchanged. Thus searching for better therapy strategies, as well as the methods to improve the efficiency of traditional therapy have become a hot topic in lung adenocarcinoma research.In recent years, small molecular antitumor compounds have attracted increasing interests in cancer therapy such as myricetin and alkaloid glycosides. These small molecular substances can either inhibit proliferation and invasion of cancer cells, or alter chemosensitivity and radiosensitivity of several malignancies, thus providing potential solutions for cancer therapy. α-Solanine, a biological active steroidal glycoside alkaloid usually derived from potato tubers or other nightshade plants, presents anti-invasion or anti-proliferation effect in several cancer types.The present researches have verified its anti-tumor function via different manners including inhibiting cell proliferation, migration and invasion, and enhance cell apoptosis, thus it’s necessary to explore the potential roles in lung cancer.The development and treatment of cancer depend on complicated molecular mechanisms in which micro RNAs(mi RNAs) are greatly involved. mi RNAs are noncoding small RNAs usually with the length of 18-24 base pairs. They can regulate the expression of the target genes by binding to 3’ UTR of m RNAs, in this way participating in a lot of biological processes, for example the invasion and migration of tumors, as well as chemosensitivity and radiosensitivity. According to recent researches, α-solanine is believed to interact with some mi RNAs and then regulate their target genes, in this way altering some properties of cancers such as cell proliferation, invasion and therapy-sensitivity.In this research we explored the anti-tumor effect in lung adenocarcinoma by two steps: firstly we evaluated the efficiency of α-solanine in inhibiting the invasion and altering chemosensitivity and radiosensitivity of lung adenocarcinoma; secondly we further explored the potential molecular mechanisms analyzing the alteration of chemosensitivity and radiosensitivity of lung adenocarcinoma.Part Ⅰ α-Solanine inhibits cell invasion and sensitizes lung adenocarcinoma cells to cisplatin and X-rayMethods1. Evaluated cell toxicity of α-solanine to human lung adenocarcinoma cell lines A549 and H1299 with MTT.2. Evaluated cell invasion and migration abilities altered by low concentration of α-solanine in A549 and H1299 cells. Tested the altered expression of invasion and migration associated genes in A549 and H1299 cells under α-solanine treatment with real-time q RT-PCR and Western blot.3. Detected the chemosensitivity change of A549 and H1299 cells by testing the inhibition of cell viability under low concentration of α-solanine and cisplatin with MTT.4. Estimated radiosensitivity change of A549 and H1299 by testing the inhibition of cell growth under low concentration of α-solanine and radiation with colony formation test.5. Carried out nude mice xeno tumor experiments to evaluate the chemosensitivity and radiosensitivity change in transplanted tumor tissues after α-solanine injection. Tested the altered expression of chemosensitivity and radiosensitivity associated genes in grafted tumor tissues under α-solanine injection with real-time q RT-PCR and Western blot.6. Statistic analysis: Using software SPSS 22.0, took LSD-t test to compare two groups and one way ANOVA for multiple groups, carried out t test to analyze measurement data; α=0.05 is considered as significance.Results1. The results of MTT tests showed that 15 μmol/L α-solanine presented obvious cell toxicity in both A549 and H1299 cells(P<0.05) while no significant cell toxicity were detected with the concentrations under 12 μmol/L.2. Transwell experiments showed that low concentration of α-solanine(3 μmol/L,6 μmol/L) could inhibit the invasion and migration of A549 and H1299 cells. Tumor invasion and migration associated genes MMP-2 and MMP-9 were significantly down-regulated, and TIMP-1 was obviously up-regulated with low concentration of α-solanine(P<0.05) according to real-time q RT-PCR and Western blot.3. The results of MTT tests and colony formation tests showed that low concentration of α-solanine could increase the ability of cisplatin and radiation to inhibit the viability of A549 and H1299 cells.4. α-Solanine significantly improved chemosensitivity and radiosensitivity in vivo in tumor transplanted mice(P<0.05). FAK, a chemosensitivity and radiosensitivity associated gene was significantly down regulated by α-solanine in grafted tumor tissue(P<0.05) according to real time q RT-PCR and Western blot.Part Ⅱ Molecular mechanism in sensitizing lung adenocarcinoma to cisplatin and X-rayMethods1 Evaluated altered FAK m RNA and protein level under low concentration of α-solanine(3 μmol/L and 6 μmol/L) in A549 and H1299 cells by real-time q RT-PCR and Western blot.2 Searched for potential mi RNAs targeting FAK with bioinformation software Target Scan and mi Randa.3 Tested altered mi R-138 expression in A549 and H1299 cells under low concentration of α-solanine(3 μmol/L and 6 μmol/L) by real-time q RT-PCR.4 Analyzed the correlation of mi R-138 and FAK m RNA expression with Pearson correlation analysis in A549 and H1299 cells.5 Amplified mutant type of FAK 3’UTR and wild type of FAK 3’UTR fragments by regular PCR or Overlap PCR, and further constructed mutant and wild type of FAK 3’UTR dual luciferase reporter vectors, co-transfected mutant or wild type vectors with either mi R-138 mimics or mi R-138 scramble into A549 cells to confirm FAK was a target gene of mi R-138.6 Transfected mi R-138 mimics or mi R-138 scramble into A549 and H1299 cells by LipofectamineTM2000, and set blank groups. Tested mi R-138 expression level in each group by real-time q RT-PCR. Evaluated the altered invasion and migration abilities of A549 and H1299 cells in each group by Transwell experiments. Added different concentrations of cisplatin to each groups, evaluated the altered chemosensitivity of cells in each group by testing cell viability with MTT test. Treated cells in different groups with radiation and carried out colony formation test in order to evaluate the altered radiosensitivity by upregulating mi R-138 expression. Evaluated altered expression of radiosensitivity and chemosensitivity associated gene FAK in each group.7 Developed FAK expression vector without 3’UTR, and transfected into A549 cells with or without mi R-138 mimics. Set blank group and 3μmol/L α-solanine groups, explored the regulation mechanism of mi R-138 on FAK with Restore experiment.8 Developed FAK specific si RNA, compared the effect of si RNA-FAK, α-solanine and mi R-138 on radiosensitivity and chemosensitivity of A549 cells.9 Statistic analysis: analyzed the results with SPSS 22.0.Results1 Low concentration of α-solanine inhibited the expression of FAK mRNA and FAK protein.2 FAK was a target gene of mi R-138 according to bioinformation analysis and further dual luciferase reporter experiment.3 Low concentration of α-solanine induced the expression of mi R-138 in A549 and H1299 cells.4 Negative correlation existed between mi R-138 and FAK m RNA expression.5 A549 and H1299 cells in mi R-138 transfection group showed decreased migration and invasion abilities compared with Scramble group and Blank group according to Transwell experiments(P < 0.05). A549 and H1299 cells in mi R-138 transfection group showed higher sensitivity to cisplatin and radiation compared with Scramble group and Blank group(P<0.05) according to MTT and colony formation tests. mi R-138 transfection group showed elevated expression of FAK compared with Scramble group and Blank group(P<0.05) according to Western blot.6 According to Restore experiment, FAK gene without 3’UTR was not regulated by mi R-138, and could restore the effect caused by mi R-138 upregulation in A549 cells.7 The effects of si RNA-FAK transfection, mi R-138 mimics and 3 μmol/L α-solanine towards A549 cells were of no significant difference, suggesting that α-solanine upregulated mi R-138 expression, and that mi R-138 further binded to 3’UTR of FAK gene and inhibited FAK gene expression, thus to improve chemosensitivity and radiosensitivity of lung adenocarcinoma.Conclusion1 Low concentration of α-solanine showed no significant influence on the viability of lung adenocarcinoma cell lines, but could inhibit the invasion and migration abilities of A549 and H1299 cells.2 Low concentration of α-solanine could upregulate mi R-138, which could bind to 3’ UTR of FAK m RNA to inhibit its expression, in this way to increase the sensitivity of lung adenocarcinoma cells to cisplatin and X-ray, thus providing a novel potential target for lung adenocarcinoma therapy.