Roles of the tumor microenvironment in the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer

Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advanced GC.In recent years,with the progress in tumor immunology research,attention has shifted toward immunotherapy as a therapeutic approach for GC.Programmed cell death protein 1(PD-1)inhibitors,as novel immunosuppressive medications,have been widely utilized in the treatment of GC.However,many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy.To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy,to maximize the clinical activity of immunosuppressive drugs,and to elicit a lasting immune response,it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients.This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment,aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.

Synergistic Effect of Hyperthermia and Neferine on Reverse Multidrug Resistance in Adriamycin-resistant SGC7901/ADM Gastric Cancer Cells

Multidrug resistance（MDR） plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine（Nef） in adriamycin（ADM） resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein（P-gp）,γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

Inhibition of apoptosis-regulatory protein Siva-1 reverses multidrug resistance in gastric cancer by targeting PCBP1

Introduction:Siva-1,as a pro-apoptotic protein,has been shown to induce extensive apoptosis in a number of different cell lines.In our previous study,we showed that overexpressed Siva-1 decreased the apoptosis of gastric cancer cells.So,we believe that it can also work as an anti-apoptotic protein.The present study aimed to determine the specific role of Siva-1 in anticancer drug resistance in gastric cancer in vivo and in vitro and preliminarily reveal the mechanism.Materials and Methods:A vincristine-resistant MKN-28/VCR gastric cancer cell line with stably downregulated Siva-1 was established.The effect of Siva-1 downregulation on chemotherapeutic drug resistance was assessed by measuring the IC50 and pump rate of doxorubicin.Proliferation,apoptosis of cells,and cell cycle were detected via colony formation assay andflow cytometry,respectively.Additionally,migration and invasion of cells was detected via wound healing and transwell assays.Moreover,we determined in vivo effects of LV-Siva-1-RNAi on tumor size,and apoptotic cells in tumor tissues were detected using TUNEL and hematoxylin and eosin staining.Results:Siva-1 downregulation reduced the pump rate of doxorubicin and enhanced the response to drug treatment.Siva-1 negatively regulated proliferation and promoted apoptosis of cells by potentiality G2-M phase arresting.Inhibition of Siva-1 expression in MKN-28/VCR cells significantly weakened wound healing ability and decreased invasion ability.Poly(C)-binding protein 1(PCBP1)was identified as a Siva-1-interacting protein in yeast two-hybrid screening.Semiquantitative RT-PCR and western blotting revealed that Siva-1 downregulation could inhibit expression of PCBP1,Akt,and NF-κB and eventually decrease the expression of MDR1 and MRP1.Conclusion:The current study demonstrated that the downregulation of Siva-1,which functions as a regulator of MDR1 and MRP1 gene expression in gastric cancer cells by inhibiting PCBP1/Akt/NF-κB signaling pathway expression,enhanced the sensitivity of gastric cancer cells to certain chemotherapies.

Gastric cancer stem cells in gastric carcinogenesis,progression,prevention and treatment

In recent decades,the study of the mechanism of tumorigenesis has brought much progress to cancer treatment.However,cancer stem cell(CSC)theory has changed previous views of tumors,and has provided a new method for treatment of cancer.The discovery of CSCs and their characteristics have contributed to understanding the molecular mechanism of tumor genesis and development,resulting in a new effective strategy for cancer treatment.Gastric CSCs(GCSCs)are the basis for the onset of gastric cancer.They may be derived from gastric stem cells in gastric tissues,or bone marrow mesenchymal stem cells.As with other stem cells,GCSCs highly express drug-resistance genes such as aldehyde dehydrogenase and multidrug resistance,which are resistant to chemotherapy and thus form the basis of drug resistance.Many specific molecular markers such as CD44 and CD133 have been used for identification and isolation of GCSCs,diagnosis and grading of gastric cancer,and research on GCSC-targeted therapy for gastric cancer.Therefore,discussion of the recent development and advancements in GCSCs will be helpful for providing novel insight into gastric cancer treatment.

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.

Non-coding RNA in drug resistance of gastric cancer

Gastric cancer(GC)is the third leading cause of cancer-related mortality worldwide.The poorly prognosis and survival of GC are due to diagnose in an advanced,non-curable stage and with a limited response to chemotherapy.The acquisition of drug resistance accounts for the majority of therapy failure of chemotherapy in GC patients.Although the mechanisms of anticancer drug resistance have been broadly studied,the regulation of these mechanisms has not been completely understood.Accumulating evidence has recently highlighted the role of non-coding RNAs(ncRNAs),including long non-coding RNAs and microRNAs,in the development and maintenance of drug resistance due to their regulatory features in specific genes involved in the chemoresistant phenotype of GC.We review the literature on ncRNAs in drug resistance of GC.This review summarizes the current knowledge about the ncRNAs’characteristics,their regulation of the genes involved in chemoresistance and their potential as targeted therapies for personalized treatment in resistant GC.

Breast cancer resistance protein(BCRP/ABCG2):its role in multidrug resistance and regulation of its gene expression

Breast cancer resistance protein(BCRP)/ATP-binding cassette subfamily G member 2(ABCG2) is an ATP-binding cassette(ABC) transporter identified as a molecular cause of multidrug resistance(MDR) in diverse cancer cells.BCRP physiologically functions as a part of a self-defense mechanism for the organism;it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract,as well as through the blood-brain,placental,and possibly blood-testis barriers.BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use.Thus,BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs.Because BCRP is also known to be a stem cell marker,its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance,self-renewal(stemness),and invasiveness(aggressiveness),and thereby impart a poor prognosis.Therefore,blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers.Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR.Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α,estrogen receptor,and peroxisome proliferator-activated receptor.Furthermore,alternative promoter usage,demethylation of the BCRP promoter,and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells.Finally,PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions.These biological events seem involved in a complicated manner.Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with cancer.This review will present a synopsis of the impact of BCRP-mediated MDR in cancer cells,and the molecular mechanisms of acquired MDR currently postulated in a variety of human cancers.

Micro RNA-145 exerts tumor-suppressive and chemoresistance lowering effects by targeting CD44 in gastric cancer

AIM To determine the potential roles of CD4 and micro RNA(mi R)-145 in gastric cancer.METHODS The levels of CD44 and mi R-145 were determined in gastric cancer cells. Quantitative real-time polymerase chain reaction was used to measure to the level of CD44 m RNA. A luciferase reporter assay and western blotting were performed to examine the effect of mi R-145 on CD44 expression. Tumor sphere and MTT assays were carried out to evaluate the self-renewal and chemo-resistance properties of gastric cancer cells.RESULTS The expression of CD44 was greatly increased and mi R-145 was decreased in gastric cancer cells that were highly enriched in cancer stem cells(CSCs). The results demonstrated that mi R-145 regulated CD44 by targeting directly the CD44 3'-untranslated region(3'-UTR). In gastric cancer cells, overexpression of mi R-145 repressed the activity of the CD44 3'-UTR, and disruption of mi R-145/CD44 3'-UTR interactions abrogated the silencing effects. In addition, mi R-145 inhibition stimulated CD44 3'-UTR activity and disruption of mi R-145/CD44 3'-UTR interactions abrogated this stimulatory effect. Enforced CD44 expression greatly increased tumor sphere formation and chemoresistance in gastric cancer cells. Furthermore, the inhibition of CSCs and the chemo-sensitivity of gastric cancer cells treated with mi R-145 were significantly abrogated by overexpression of CD44. CONCLUSION mi R-145 targeting of CD44 plays critical roles in the regulation of tumor growth and chemo-resistance in gastric cancer.

The expression and significance of the multidrug resistance-related proteins P-gp, MRP and LRP in human non-small cell lung cancer tissues

Objective: To explore the expression and significance of the multidrug resistance-related proteins P-glycopro-tein (P-gp), multidrug resistance-related protein (MRP), lung resistance protein (LRP) in human non-small cell lung cancer (NSCLC) tissues and paratumor tissues. Methods: Immunohistochemistry (IHC) was used to examine the expression level of proteins P-gp, MRP and LRP in 43 samples of NSCLC and 15 samples of paratumor tissues. Results: The expression rates of P-gp, MRP and LRP in 43 tumor tissues were 74.42% (32/43), 67.44% (29/43) and 88.37% (38/43), respectively, while in 15 paratumor tissues were 13.33% (2/15), 20.00% (3/15) and 6.67% (1/15), respectively. There was significant difference in the expression of proteins (P-gp, MRP and LRP) between lung cancer tissues and paratumor tissues (P < 0.05). The expres-sion of proteins P-gp, LRP in lung adenocarcinoma were higher than that in other pathological carcinomas (P < 0.05). The expression of protein MRP was not related to pathological type, clinical stage and classification of histodifferentiation (P > 0.05). Conclusion: Multidrug resistance is more common in NSCLC. The proteins of P-gp, MRP and LRP participated in the formation of multidrug resistance in lung cancer. Detection of multidrug resistance-related proteins in lung cancer tissues may be useful to choice drugs.

EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN IN HUMAN GASTRIC AND RENAL CARCINOMAS

Objective The clinical signilicance of exPression of multidrug resistance- associated protein (MRP) in gastric and renal carcinoma was investigated. Methods LSAB immunohistochemistry was performed to detect eopression of MRP in the carcinoma tissues of 52 patients with gastric carcinoma and 20 cases with renal cell carcinoma. Results The positive expression rate of MRP was 38.5% (20/52) in gastric carcinoma tissues, and 60% (12/20) in renal carcinoma tissues. The expression of MRP both on cellular membrane and in cytoplasm was observed, but the expression in cytoplasm (thick granule) was more obvious. The positive expression rates of MRP in advanced gastric and renal carcinoma (Ⅲ orⅣ stage) were 60% (15/25) and 88.90% (8/9) reSPectively, which were higher than those in early lesion (Ⅰ or Ⅱ stage, 18.5% and 36.4% respectively). Furthermore, the patients with positive expression of MRP in gastric carcinoma tissues had shorter mean survival time and lower 5-year survival rate than that with negative eopression of MRP. Conclusion MRP plays an important role in the infiltration and metastasis of gastric and renal carcinoma and might contribute to the intrinsic drug - resistance in both carcinomas.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

EXPRESSION OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) AND ITS RELATIONSHIP WITH CLINICOPATHOLOGICAL FACTORS IN NON-SMALL CELL LUNG CANCER

Objective: To investigate the relationship between the expression of multidrug resistance-associated protein (MRP) and clinicopathological factors and prognosis. Methods: The expression of MRP in 62 cases with non-small cell lung cancer (NSCLC) was detected using immunohistochemistry method. The expression of MRP in 30 cases of NSCLC and corresponding normal lung tissues were detected using immunohistochemistry and Western Blot. Results: this study of tumor tissues confirmed the plasma membrane and/or cytoplasm locations of MRP. There was apparent difference between normal lung tissues and NSCLC in MRP. The survival analysis of 62 NSCLC showed that the mean survival time of the patients with negative MRP expression was 69.8117.41 months and that of patients with positive MRP expression, 25.384.46 months. Log-rank test suggested that the difference between them was significant (P=0.0156). It was also found that in squamous cell lung cancer the statistically significant difference between the mean survival time of patients with positive MRP expression and those with negative MRP expression (P=0.0153). Multivariate Cox model analysis suggested that the survival time was significantly related to expression of MRP (P=0.035) and lymphatic metastasis (P=0.038). Conclusion: MRP expression in NSCLC is significantly higher compared with normal lung tissues. The mean survival time of patients with negative MRP was relative longer and expression of MRP was an independent factor for prognosis.

Characterization of cancer stem-like cells in a novel STI571-resistant chronic myeloid leukemia cell line

Objective： To characterize a novel chronic myeloid leukemia （CML） cell line and to further elucidate the mechanisms of resistance to STI571. Methods： A novel K562 cell line （K562NP16） was achieved after exposure of the K562 cells to VP16. A small subpopulation （K562NP16 SP） that was capable of excluding Hoechst 33342 in the K562NP16 cell line was isolated by fiow cytometry sorting. The rest of the K562NP16 cells were classified as non-SP K562NP16. The mechanisms involved in K562NP16 SP cells which became resistant to STI571 were studied. Results： The levels of Bcr-Abl and Abl proteins were similar in the K562 cell line and in non-SP K562NP16 and K562NP16 SP cells. The multidrug-resistant gene 1 （MDR1） expression of the 170 kDa P-glycoprotein （P-gp） was detected in K562NP16 non-SP and K562NP16 SP cells but not in K562 cells. The expression levels of P-gp in the two K562NP16 cell lines were similar. Compared with non-SP K562/ VP16, the K562NP16 SP cells were more resistant to STI571. This resistance could hardly be reversed by many multidrug resistance inhibitors. In addition, in vivo study showed that the K562NP16 SP cells induced tumorigenesis in mice, while the K562NP16 non-SP cells failed to do so. Conclusion： A novel K562 cell line, K562NP16, was generated. A small side population K562NP16 SP cells, had high resistance to STI571 treatment and more tumorigenic than the K562 cells. It may represent the cancer stem cells of the K562NP16 cell line.

EXPRESSION OF BCRP GENE IN THE NORMAL LUNG TISSUE AND LUNG CANCER

Objective: To investigate the expression of novel multidrug resistance transporter (BCRP gene) from human MCF-7/AdrVp breast cancer cells in normal lung tissue and non-small lung cancer tissue. Methods: RNA was extracted immediately from fresh normal lung tissue and viable tumor tissue harvested from surgically resected specimens of non-small cell lung cancer patients. cDNA of BCRP gene was prepared by RT-PCR and was then amplified by PCR. cDNA products from those specimens were transferred to blotting membrane through electrophoresis and transferring technique and southern blot hybridization was eventually performed to detect the expression of BCRP gene. Results: RNA were extracted from 8 tumor tissue alone and 12 pairs of tumor tissue and normal lung tissue harvested from the same lung. Four patients’ RNA samples with poor quality due to degrading were discarded. cDNA products of BCRP gene were obtained by RT-PCR and were then amplified by PCR in the remain 16 patients’ RNA samples. Through southern blot hybridization, BCRP gene was found to be slightly expressed in various amounts in all normal lung tissue (10/10) and only in a half of tumor tissue samples (8/16). Conclusion: BCRP gene is slightly expressed in different amount in all normal lung tissue and only in a half of tumor tissue of non-small cell lung cancer patients. It is possible to induce it’s overexpression and to develop multidrug resistance during chemotherapy if using anthracycline anticancer drugs.

Expression of ABCG2 in human gastric carcinoma

Objective:The aim of this study was to investigate the expression of ABCG2 in human gastric carcinoma and its clinical significance.Methods:Expression of ABCG2 was examined with immunohistochemical technique in the specimens from 45 gastric carcinoma tissues and 30 surrounding normal tissues.The mRNA expression of ABCG2 was measured by RT-PCR and real-time quantitative PCR in 30 cases of gastric carcinoma and normal gastric mucosa, respectively.Results:ABCG2 expression was observed in 28 of 45(62.2%) cases by immunohistochemical analysis.In ABCG2-positive tumors, adjacent non-neoplastic tissue was similarly analyzed, revealed that ABCG2 was up-regulated in gastric carcinoma.ABCG2 expression in poorly differentiated/undifferentiated carcinoma was significantly higher than that in well/moderately-differentiated carcinoma(P < 0.05).The mRNA expression of ABCG2 was significantly higher than that in normal gastric mucosa(P < 0.05).Conclusion:ABCG2 plays an important role in the multi-drug resistance of gastric carcinoma.ABCG2 might be an important factor in the research of gastric cancer stem cell.

小细胞肺癌耐药细胞的建立及生物学特性和多药耐药性鉴定

目的建立对顺铂与依托泊苷抵抗的小细胞肺癌(small cell lung cancer,SCLC)耐药细胞NCI-H446/EP,对其生物学特性和多药耐药性进行评价。方法裸鼠皮下接种SCLC NCI-H446细胞构建异种移植瘤体内模型,给予SCLC一线EP方案治疗,诱导体内耐药,剥离瘤组织体外培养获得耐药细胞。体外实验检测其耐药系数,细胞倍增时间,细胞周期分布,多药耐药基因(MDR1)、耐药相关蛋白的表达,体内验证其耐药性。结果NCI-H446成瘤裸鼠给予EP治疗,8周后产生获得性耐药,经分离培养获得耐药细胞株NCI-H446/EP。该细胞株对顺铂、依托泊苷、SN38和阿霉素的耐药系数分别是12.01、18.36、65.4和10.12。与亲本细胞相比,耐药细胞G 0/G 1期比例明显增加,G 2/M期细胞比例减少,倍增时间延长。耐药细胞MDR1在mRNA及蛋白水平均高于亲本细胞,在体内也具有耐药性。结论成功构建了SCLC耐药细胞株NCI-H446/EP,该细胞具有多药耐药的基本特征,可用于抗SCLC药物筛选及耐药机制的研究。

Sorcin高表达与胃癌细胞株SGC7901多药耐药的关系

背景与目的:长春新碱耐药胃癌细胞SGC7901/VCR是具有P-糖蛋白(P-gp)高表达的典型多药耐药细胞,但P-gp抑制剂维拉帕米(VRP)不能完全逆转其耐药,说明还存在其它耐药机制。我们运用差异蛋白质组学技术研究发现,可溶性耐药相关钙结合蛋白(Sorcin)在SGC7901/VCR中表达较SGC7901细胞明显上调。为进一步阐明Sorcin与胃癌细胞耐药的关系,本研究构建了FLAG-Sorcin融合表达载体,探讨Sorcin在胃癌多药耐药中的作用。方法:采用逆转录聚合酶链反应法(reverse transcription-polymerase chain reaction,RT-PCR)扩增Sorcin cDNA片段编码区序列全长,DNA重组技术构建FLAG-Sorcin融合表达载体,经脂质体介导稳定转染SGC7901细胞,RT-PCR及Western blot分别检测转染细胞Sorcin mRNA表达的变化;噻唑蓝(MTT)法测定转染细胞及对照细胞对化疗药物的敏感性。用Sorcin反义核苷酸转染SGC7901-F-SOR细胞,RT-PCR检测Sorcin表达水平的变化,MTT法测定转染后细胞对VCR敏感性的变化。结果:RT-PCR法扩增出616bp全长Sorcin cDNA片段,成功构建了FLAG-Sorcin融合表达载体;RT-PCR及Western blot证实,Sorcin在SGC7901转染细胞中高表达;Sorcin高表达的SGC7901细胞对长春新碱(VCR)、阿霉素(ADM)、紫杉醇(Taxol)、氟尿嘧啶(5-FU)分别产生了8.87倍、6.13倍、6.67倍和2.80倍耐药。Sorcin反义核苷酸转染SGC7901-F-SOR细胞后,RT-PCR验证Sorcin表达被抑制,MTT结果显示细胞对VCR敏感性增加。结论:Sorcin高表达可致SGC7901细胞产生低倍数的多药耐药,说明Sorcin与SGC7901细胞多药耐药密切相关,有望成为逆转胃癌多药耐药的靶点。

华蟾素对HepG_(2)/ADM细胞耐药的抑制作用及其机制研究

目的观察华蟾素对人肝癌细胞HepG_(2)/阿霉素(ADM)耐药性的影响并分析其可能的作用机制。方法用不同浓度的华蟾素处理HepG_(2)和HepG_(2)/ADM细胞24 h,MTT法检测细胞增殖抑制率,计算半数抑制浓度(IC 50);倒置显微镜观察细胞形态学变化;药物蓄积实验检测对细胞内药物累积的影响;Western blot实验检测凋亡相关蛋白Bcl-2、Bax、Caspase-9、Caspase-3表达情况。结果华蟾素能对HepG_(2)和HepG_(2)/ADM细胞具有增殖抑制作用,耐药倍数为4.67;随华蟾素处理浓度升高,HepG_(2)/ADM细胞体积变小,细胞皱缩突起减少,细胞间连接减少;与耐药组比较,华蟾素可提高HepG_(2)/ADM细胞对阿霉素的蓄积水平,上调Bax、Caspase-9、Caspase-3蛋白表达(P<0.05),下调Bcl-2蛋白表达(P<0.01),促进细胞凋亡。结论华蟾素能够显著抑制HepG_(2)/ADM细胞增殖,增加ADM在细胞内的蓄积,其作用机制可能与调控Bcl-2/Bax蛋白表达,诱导细胞凋亡有关。

Integrin β1对胃癌细胞SGC7901多药耐药性的影响

目的:探究整合素β1(integrinβ1)对胃癌多药耐药性的影响及可能的作用机制。方法:Western blot法及q PCR实验检测胃癌细胞株SGC-7901及胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达情况。采用integrinβ1反义寡核苷酸转染,敲减胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot法检测integrinβ1、Bcl-2/Bax、cleaved caspase-3/caspase-3、细胞色素C(CytC)和p-AKT/AKT的蛋白水平。结果:耐药细胞株SGC7901/DDP中integrinβ1的mRNA及蛋白表达水平均明显高于亲本细胞株;并且在亲本细胞株SGC7901中加入顺铂、长春新碱及5-氟尿嘧啶等化疗药物刺激后,integrinβ1的蛋白表达水平明显升高。敲减integrinβ1的表达可诱导胃癌耐药细胞SGC7901/DDP的凋亡,增加细胞对化疗药物的敏感性;此外下调Bcl-2/Bax、p-AKT^(Ser473)和p-AKT^(Thr308)的蛋白水平,同时促进线粒体Cyt-C的释放,上调cleaved caspase-3的蛋白水平。结论:敲减胃癌顺铂耐药细胞SGC7901/DDP的integrinβ1表达可恢复细胞对化疗药物的敏感性,促进细胞经线粒体路径的凋亡,其机制可能与抑制AKT的磷酸化,阻断该信号通路有关。

EP联合方案耐药小细胞肺癌细胞株的构建及机制探讨

目的构建依托泊苷(etoposide,VP-16)联合顺铂(cisplatin,DDP)化疗方案(EP联合方案)耐药的小细胞肺癌(small cell lung cancer,SCLC)细胞株H446/EP,并进行耐药特性鉴定及机制探讨。方法以药物浓度递增法,使用VP-16联合DDP处理NCI-H446细胞构建H446/EP细胞株。以H446/EP与NCI-H446细胞为研究对象,MTT法检测细胞活力,计算H446/EP细胞耐药指数(resistance index,RI);细胞克隆实验、Incucyte细胞增殖(无标记)法检测细胞增殖能力;转录组学测序后,对2种细胞差异表达基因进行富集分析;Western blot检测细胞耐药、DNA损伤修复(repair of DNA damage,DDR)、自噬标志分子的蛋白表达量。结果MTT结果显示,H446/EP细胞对VP-16、DDP与DOX的RI分别为6.14、3.43与1.96;细胞克隆实验与Incucyte细胞增殖(无标记)法结果显示,H446/EP细胞增殖能力显著高于NCI-H446细胞(P<0.01);转录组学测序、通路富集分析显示差异表达基因在肿瘤化疗耐药、DDR、自噬相关通路富集;Western blot结果显示,相比NCI-H446细胞,H446/EP细胞MRP1、BCRP、RAD51、γ-H2AX及LC3-II/LC3-I蛋白表达量显著升高,p62蛋白表达量显著降低(P<0.05)。结论EP联合方案耐药的SCLC细胞株H446/EP构建成功,增殖能力增强;细胞外排转运蛋白表达量增加、DDR及自噬水平升高可能是SCLC对EP联合方案产生耐药的机制。