Geranylgeranyl transferase 1 inhibitor GGTI­298 enhances the anticancer effect of gefitinib.

Dysregulation of epidermal growth factor receptor (EGFR) signaling is responsible for the resistance to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and is thereby associated with the progression of tumors in non­small cell lung cancers (NSCLCs). Immunoblotting results revealed that geranylgeranyl transferase 1 inhibitor (GGTI)­298, a geranylgeranyl transferase 1 inhibitor with potential antitumor effects, effectively inhibited the phosphorylation of EGFR and its downstream target protein kinase B (AKT). A combination of gefitinib and GGTI­298 amplified the inhibition of the EGFR­AKT signaling pathway. In addition, GGTI­298 treatment produced a synergistic effect on the inhibition of proliferation as indicated by the combination index values of <1 when combined with gefitinib in the NSCLC cell lines HCC827 and A549. These synergistic effects were also observed to induce apoptosis and migration inhibition. Further mechanistic studies demonstrated that GGTI­298 inhibited the activity of Ras homolog family member A (RhoA), and downregulation of RhoA with small interfering RNA impaired the phosphorylation of EGFR, which suggested that EGFR inhibition by GGTI­298 may be exerted mainly through RhoA mediation. These results presented a novel, promising therapeutic strategy involving a combination of two drugs for targeting EGFR signaling in lung cancer.

Inhibition of YAP reverses primary resistance to EGFR inhibitors in colorectal cancer cells.

Mutant KRAS and BRAF are associated with primary EGFR inhibitor resistance in colorectal cancer (CRC). However, other biomarkers that could predict EGFR inhibitor resistance remain elusive. In the present study, immunoblotting and cell proliferation results revealed that yes­associated protein (YAP), a downstream effector of the Hippo pathway, was positively associated with primary cetuximab resistance in CRC cells. YAP knockdown enhanced the cytotoxicity of cetuximab in CRC cells. Simvastatin, a 3­hydroxy­3­methylglutaryl­coenzyme A (HMG­CoA) reductase inhibitor of the mevalonate pathway that inhibits YAP bioactivity through nuclear translocation and total YAP expression, increased the cytotoxicity of EGFR inhibitors (cetuximab and gefitinib) against CRC cells. The combination of simvastatin and EGFR inhibitors inhibited YAP and EGFR signaling more markedly than each agent alone. Adding back geranylgeranyl pyrophosphate (GGPP), a key product of the mevalonate pathway, reversed the YAP bioactivity inhibition induced by simvastatin and the cell proliferation inhibition induced by the combination of simvastatin and EGFR inhibitors. Collectively, these results revealed that YAP may be useful in identifying cetuximab resistance in CRC and indicated that targeting of both YAP and EGFR signals may present a promising therapeutic approach for CRC.

Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay.

Deregulated activity of Ras GTPases has been observed in many types of human cancers, and contributes to the diverse aspects of carcinogenesis. Although the significance in tumorigenesis has been widely accepted and many therapeutic drugs are under development, little attention has been dedicated to the development of sensors for the Ras activity in vivo. Therefore, based on the split firefly luciferase complementation strategy, we developed a monomolecular bioluminescent biosensor to image endogenous Ras activity in living subject. In this biosensor, two inactive luciferase fragments are sandwiched by Raf-1, whose conformation changes upon GTP-Ras binding. Thus, the Ras activity can be surrogated by the intensity of the complementary luciferase. The bioluminescence analyses demonstrated that this novel biosensor behaved the robust and sensitive reporting efficiency in response to the dynamical changes of Ras activity, both in living colorectal cancer cells and in vivo. Compared to the traditional method, such as the pull-down assay, the bioluminescent sensor is simply, noninvasive, faster and more sensitive for the analysis of the endogenous Ras activity. This innovative work opens up the way for monitoring the preclinical curative effect and high-throughput screening of therapeutic drugs targeting Ras pathways.

[The effect of eukaryotic expression plasmid ARHI on gastric cancer].

OBJECTIVE: To study the effect of expressed aplasia ras homolog member I (ARHI) on the malignant biological behaviors of gastric cancer including the proliferation, migration and invasion of the cell. METHODS: The eukaryotic expression plasmid of ARHI was constructed and transfected into MKN-28 cell with lipofectamine 2000 as pEGFP-ARHI group, transfected with pEGFP-N1 as pEGFP-N1 group, and untreated MKN-28 as control group. The expression of ARHI was detected by Western blotting and fluorescence microscope. CCK-8 assay was used to analyze the cell proliferation, the wound-healing assay and transwell assay were performed to investigate the effects on migration and invasion. RESULTS: Compared with the pEGFP-N1 group and control group, proliferation, invasion and migration of the pEGFP-ARHI group were depressed (P < 0.05). CONCLUSION: Recombination eukaryotic expression pEGFP-ARHI could partially reverse the malignant phenotypes of gastric cancer cell MKN-28.

Serum miR-19a predicts resistance to FOLFOX chemotherapy in advanced colorectal cancer cases.

BACKGROUND: Colorectal cancer is the fourth most common cancer worldwide and the second leading cause of cancer-related death. FOLFOX is the most common regimen used in the first-line chemotherapy in advanced colorectal cancer, but only half of the patients respond to this regimen and we have almost no clue in predicting resistance in such first-line application. METHODS: To explore the potential molecular biomarkers predicting the resistance of FOLFOX regimen as the first-line treatment in advanced colorectal cancer, we screened microRNAs in serum samples from drug-responsive and drug-resistant patients by microarrays. Then differential microRNA expression was further validated in an independent population by reverse transcription and quantitative real- time PCR. RESULTS: 62 microRNAs expressing differentially with fold-change >2 were screened out by microarray analysis. Among them, 5 (miR-221, miR-222, miR-122, miR-19a, miR-144) were chosen for further validation in an independent population (N=72). Our results indicated serum miR-19a to be significantly up-regulated in resistance-phase serum (p=0.009). The ROC curve analysis showed that the sensitivity of serum miR-19a to discriminate the resistant patients from the response ones was 66.7%, and the specificity was 63.9% when the AUC was 0.679. We additionally observed serum miR-19a had a complementary value for cancer embryonic antigen (CEA). Stratified analysis further revealed that serum miR-19a predicted both intrinsic and acquired drug resistance. CONCLUSIONS: Our findings confirmed aberrant expression of serum miR-19a in FOLFOX chemotherapy resistance patients, suggesting serum miR-19a could be a potential molecular biomarker for predicting and monitoring resistance to first-line FOLFOX chemotherapy regimens in advanced colorectal cancer patients.

Chemotherapy and radiotherapy downregulate the activity and expression of DNA methyltransferase and enhance Bcl-2/E1B-19-kDa interacting protein-3-induced apoptosis in human colorectal cancer cells.

Bcl-2/E1B 19-kDa interacting protein 3 (BNIP3) is a proapoptotic protein whose expression level is often low in colorectal cancer (CRC) cells due to the BNIP3 gene promoter DNA methylation by DNA methyltransferase (DNMT). It is known that chemotherapy and radiotherapy suppress CRC through inducing tumor apoptosis. However, the molecular mechanisms underlying chemotherapy and radiotherapy-induced apoptosis of CRC cells are not well defined. In this study, we observed that the expression level of BNIP3 in colon cancer cells was significantly increased by treatment with therapeutic agents and radiation in vitro. The BNIP3 protein level in CRC tissues from patients who received preoperative concurrent chemotherapy was significantly higher than in those who received surgery alone. Furthermore, treatment with chemotherapeutic agents and radiation significantly decreased the DNMT1 expression level and enzymatic activity. Both expression level and activity of DNMT1 were inversely correlated with the expression level of BNIP3 in colon carcinoma cells after treatment with chemotherapeutic agents and radiation. Consistent with increased BNIP3 expression, chemotherapeutic agents and radiation induced colon carcinoma cell apoptosis in a dose-dependent manner. Based on these observations, we conclude that chemotherapy and radiotherapy inhibit DNMT1 expression to upregulate BNIP3 expression to promote CRC cell apoptosis. And, BNIP3 may play a role in the caspase-dependent apoptosis pathways, mainly during treatment with chemotherapy and radiotherapy.

[The effect of RhoE on CD44 promoter and the malignant behaviors of colorectal cancer cell].

OBJECTIVE: To study the effect of RhoE on the transcriptional regulation of cd44 and in vivo tumorigenicity of nude mice. METHODS: cd44 promoter was amplified from human embryonic kidney HEK293 cells with PCR and insert into Dual-Luciferase Reporter plasmid pGL3-Basic. After confirmed with sequence analysis, the generated recombinant was transfected into SW480 and LoVo cells to monitor their activity. Colon cancer SW480 and LoVo cells were cotransfected with pGL3-CD44 promoter along with pcDNA3. 1-RhoE and pcDNA3. 1 respectively. SW480 and LoVo cells were stably transfected with pcDNA3. 1-RhoE and the control group and were inoculated into nude mice to observe tumor growth. Immunohistochemistry assay was applied to observe the morphology of tumor cells and the expression of CD44 molecules. RESULTS: The cd44 promoter sequence was amplified correctly, Dual-Luciferase Reporter Assay showed that the constructed reporter gene has promoter activity. The expression of cd44 promoter sequence containing reporter gene in pcDNA3. 1-RhoE expression positive LoVo cells was inhibited; HE staining demonstrated that the pcDNA3. 1-RhoE transfected tumor cells was significantly smaller than that in the control group, and consistent size and shape tumor cells were observed but no tumor giant cells, the corresponding volume of the tumor nuclei were also small. CONCLUSION: RhoE could partially reverse the malignant biological behavior of tumors by inhibiting the transcriptional regulation of cd44 promoter.

[The effect of RhoA and proteasome inhibitor MG132 on angiogenesis in tumors].

OBJECTIVE: To investigate the effect of RhoA to VEGF, HIF-1alpha and MVD (microvascular density) and the effect of MG132 to RhoA. METHODS: The constitutively-active mutant vectors of RhoA (pCEFL-GST-V14RhoA) were transfected into gastric cancer cell line MKN-45 by Lipofectamine 2000, single clones were selected by G418 and identified with western blot. The content of VEGF in the conditioned media was detected by ELISA. Constitutively-active RhoA nude mice models were established and treated with MG132. The effect of RhoA and MG132 on expression of HIF-1alpha, VEGF and CD31 were detected by immunohistochemistry. RESULTS: Cell line of stable-transfected constitutively-active RhoA was established and constitutively-active RhoA could stimulate secretion of VEGF but MG132 inhibited that. Constitutively-active RhoA could obviously induce growth of tumor (P < 0.05), but MG132 inhibited it (P < 0.05). Constitutively-active RhoA could promote protein of HIF-1alpha, VEGF and CD31 but MG132 inhibited the function of RhoA (P < 0.05). CONCLUSION: Our studies indicates that MG132 could affect angiogenesis of tumors through inhibition the regulating function of RhoA on HIF-1alpha, VEGF and CD31.

[The affect of Si-Rac1b on the malignant biological behaviors of colorectal cancer cell].

OBJECTIVE: To observe the affect of Small interfering RNA Rac1b (Si-Rac1b) on the malignant biological behaviors of colorectal cancer cell including the proliferation, migration, invasion and apoptosis of the cells. METHODS: Mediated by lipofectamine 2000, Si-Rac1b was transfected into colorectal cancer cell line SW1116 (with overexpression of Rac1b). The expression of Rac1b was detected by Western blotting and RT-PCR. The CCK-8 assay was used to analyze the cell proliferation, the Wound-healing assay and invasion assay were respectively applied to analyze the cell migration and invasion, and the Hoechst 33258 was used to evaluate the apoptotic index. RESULTS: Si-Rac1b can knock down the Rac1b but not Rac1 both in the level of mRNA and protein. In addition, Si-Rac1b could singnificantly facilitate the cell proliferation, migration, invasion and control the cell apoptosis. CONCLUSION: Si-Rac1b could partically reverse the malignant phenotypes of colorectal cancer cell.

[The effect of small GTPase Cdc42 on the multidrug resistance of oxaliplatin-resistant colon cancer cell lines].

OBJECTIVE: To determine the effect of cell division cycle42 (Cdc42) on the multidrug resistance of oxaliplatin-resistant human colon cancer cells. METHODS: The protein expression levels of Cdc42 in oxaliplatin-resistant colon cancer cells and parental cells were examined with Western blot. pDEST26-His-Cdc42 was transfected by lipofectamine 2000 into SW480 and Colo320 cells with low expression of Cdc42. Cdc42 siRNA was transfected by lipofectamine 2000 into SW480/L-OHP and Colo320/L-OHP cells with high expression of Cdc42. The expression of Cdc42 in these cell lines were examined by Western blot and RT-PCR 48 hours after transfection. The sensitivity of colon cancer cells to antitumor drugs was evaluated using CCK-8 assay. The concentration of each drug that caused a 50% reduction in the numbers of cells (IC50) was calculated. The expression of P-gp, MRP1 in SW480/Cdc42 and Colo320/ Cdc42 cells were examined with Western blot. RESULTS: Cdc42 was over-expressed in the SW480/L-OHP and Colo320/L-OHP cell lines. Over-expression of Cdc42 significantly enhanced the resistance of colon cancer cells to multiple antitumor drugs and up-regulated the expression of P-gp and MRP1. CONCLUSION: Cdc42 enhances the resistance of colon cancer cells to several antitumor drugs. It might become a potential target for reversing multidrug resistance of colon cancer.

Effects of the proapoptotic regulator Bcl2/adenovirus EIB 19 kDa-interacting protein 3 on radiosensitivity of cervical cancer.

PURPOSE: Bcl2/adenovirus EIB 19 kDa-interacting protein 3 (BNIP3) is a proapoptotic member of the Bcl-2 family. To address its potential as a therapeutic target for radiosensitization, this study investigated the effect of Bnip3 expression on radiosensitivity of cervical cancer in vitro and in vivo. MATERIALS AND METHODS: In vitro: A plasmid expressing the BNIP3 gene was transfected into human cervical cancer HeLa cells using Lipofectamine(2000), and western blot and immunohistochemistry analysis were performed to evaluate the expression of BNIP3 in transfected cells. The effects on radiation-induced apoptosis were investigated using a clone formation assay and flow cytometry. In vivo: A total of 6 × 106 HeLa cells were subcutaneously inoculated into the dorsal flank of nude mice, and plasmids expressing the BNIP3 gene were injected into the mice via the tail vein. Tumor volume was calculated, and immunohistochemistry was used to detect the expression of BNIP3 in tumor cells. TUNEL assays were performed to determine the apoptosis rates in tumor tissues. RESULTS: Transfection with the recombinant BNIP3 plasmid increased expression of the Bnip3 protein in tumor cells. This apoptosis regulator significantly decreased the viability of cells (p < 0.01) and increased the apoptosis rates (p < 0.01) both in vitro and in vivo. The antitumor effect of radiotherapy was enhanced by overexpression of BNIP3, as revealed by tumor growth curve analysis. CONCLUSIONS: Radiosensitization in human cervical cancer cells was observed after treatment with the recombinant BNIP3 plasmid in vitro and in vivo. Results suggested that BNIP3 may play a role in enhancement of radiotherapy efficiency, and its expression may have a synergistic effect on radiation treatments.

[Analysis of gene expression patterns in an irinotecan-resistance colon cancer cell by cDNA microarray].

OBJECTIVE: To study the mechanism of irinotecan-resistant colon cancer by analyzing the differential gene expression patterns with cDNA microarray. METHODS: Total RNA was purified from irinotecan-sensitive colonic cancer cell line, SW480 and its irinotecan-resistance cell line, SW480/CPT. The cRNA retro-transcribed from RNA were labeled with Cy3 fluorescence as probes. The probes were hybridized with Agilent gene chips and the fluorescence images of the chips were obtained with Axon 4000B scanner as well as analyzed with Genepix 3.0 software. The microarray results were confirmed by reverse transcription-polymerase chain reaction. RESULTS: Of the 1598 genes with altered expressions, there were 911 up-regulated genes and 687 down-regulated genes. Glutathione S-Transferase (GST) isoenzyme family GSTA such as GSTA1, GSTA2, GSTA3 and GSTA5 were significantly up-regulated. The expression levels of many Zinc finger protein family members (ZNF) were also differentially regulated. CONCLUSION: GSTA and ZNF subunit genes might play an important regulation role in the irinotecan resistance of colon cancer.

Establishment and biological characteristics of oxaliplatin-resistant human colon cancer cell lines.

BACKGROUND AND OBJECTIVE: Chemotherapy is the main treatment for colon cancer, while multidrug-resistance is the main reason for chemotherapy failure and tumor relapse. This study was to establish two oxaliplatin-resistant colon cancer cell lines and evaluate their biological characteristics. METHODS: Oxaliplatin-resistant colon cancer cell lines SW620/L-OHP and lovo/L-OHP were established in vitro by continuous exposure to oxaliplatin (L-OHP) of low and gradually increased concentration. Growth curve, cross-resistance and resistance index of the oxaliplatin-resistant cell lines to various anti-cancer agents were determined by CCK8 assay. The expressions of P-glycoprotein (P-gp), multidrug-resistance protein 1 (MRP1) and MRP2 were detected by Western blot. Cell cycle distribution as well as the expression of CD133 and CD44 were measured by flow cytometry. RESULTS: It took 10 months to establish the SW620/L-OHP and LoVo/L-OHP cell lines with stable resistance to oxaliplatin. Cross-resistance to 5-fluorouracil, etoposide, cisplatin, vincristine and epirubicin but not to paclitaxel was observed. Longer doubling time, higher proportion of cells in G(0)/G(1) phase and lower proportion in G(2)/M phase were observed in the two oxaliplatin-resistant cell lines compared with their parental cell lines. The expression of MRP2 in the oxaliplatin-resistant cells was up-regulated, while those of P-gp and MRP1 had no significant change. CD133 was overexpressed while CD44 level remained unchanged in SW620/L-OHP and LoVo/L-OHP cells. CONCLUSIONS: SW620/L-OHP and LoVo/L-OHP cell lines show a typical and stably resistant phenotype and may be used as research models.

Effects of microRNA-29 family members on proliferation and invasion of gastric cancer cell lines.

BACKGROUND AND OBJECTIVE: MicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells. METHODS: We detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion. RESULTS: Similar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05). CONCLUSIONS: Members of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.

[Effect and mechanism of DNMT inhibitor on the reversal of multidrug resistance in human colon cancer cell line sw620/L-OHP].

OBJECTIVE: To explore whether demethylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could reverse multidrug resistance (MDR) of human colon cancer and its impacts on the expressions of Bcl-2/adenovirus E1B 19 KDa protein interacting protein 3 (BNIP3), P-glycoprotein (P-GP) and multidrug resistance-associated protein (MRP), as well as the activity of DNA methyltransferase (DNMT). METHODS: Oxaliplatin (L-OHP) resistant human colon carcinoma cells (sw620/L-OHP) and its parental cells (sw620) were used to determine the effect of 5-aza-dC. CCK-8 assay was adopted to evaluate the cytotoxicity of 5-aza-dC. Western blot was performed to observe the expressions of BNIP3, P-GP and MRP. H microassay was used to detect the activity of DNMT. RESULTS: After the addition of 1.4 micromol/L 5-aza-dC, the 50% inhibitory concentration (IC50) of L-OHP to sw620/ L-OHP cells was decreased from (0.335 +/- 0.043) microg/mL to (0.069 +/- 0.023) microg/mL, and the chemosensitivity increased 8.26 times (P < 0.001). Western blot analysis showed that the expression of BNIP3 was up-regulated by 5-aza-dC in a concentration-dependent manner (P < 0.001), and P-GP and MRP expression were also significantly changed (P < 0.05) but not in a concentration-dependent manner (P > 0.05). DNMT activity was down-regulated by 5-aza-dC in sw620/L-OHP cells (P < 0.001). CONCLUSIONS: 5-aza-dC could reversed multi drug resistance of sw620/L-OHP, which may due to the demethylation to upregulate BNIP3 expression, and inhibit the expression of resistance-associated protein P-GP and MRP further by some unknown mechanism.

[The effect of Rac1 siRNA on the biological behaviors of gastrointestinal cancer cells].

OBJECTIVE: To explore the effect of Rac1 siRNA on the expression of Rac1 and the biological behaviors of gastrointestinal cancer cells. METHODS: Rac1 siRNA was transfected into human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo by lipofectamine 2000. The expression of Rac1 in these cell lines were detected by Western blot and RT-PCR after 48 hours of the transfection. The effect of Rac1 on the proliferation of human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo were tested by CCK8 kit. The motility of the transfected and the control cancer cells were assessed by Wound-healing assay and invasion assay. The apoptotic index was evaluated by Hoechst 33258 staining and FCM. RESULTS: Rac1 siRNA can down-regulated the expression of Rac1 on human gastric cancer cell line SGC803 and colorectal cancer cell line Lovo remarkably, and Rac1 siRNA can inhibit both the proliferation and motility of the transfectants. Analysis of apoptosis demonstrated that Rac1 siRNA can promote apoptosis of the gastric cancer cells and colorectal cancer cells. CONCLUSION: Rac1 play an important role in the regulation of biological behaviors of human gastric cancer and colorectal cancer cells, and the interference of Rac1 expression could provide a novel path in reversing the malignant phenotypes of these malignancies.