Impact of RNA interference targeting hypoxia-inducible factor-1alpha on chemosensitivity in esophageal squamous cell carcinoma cells under hypoxia
Department of Oncology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. Zhonghua yi xue za zhi
To investigate the impact of RNA interference (RNAi) targeting hypoxia-inducible factor 1alpha (HIF-1 alpha) on chemosensitivity of esophageal squamous cell carcinoma cells under hypoxia.
Human esophageal squamous cell carcinoma cells of the line EC9706 were cultured and divided into 3 groups: untransfected group, added with cobalt chloride (CoCl(2)), a chemical hypoxia inducer, for 8 h so as to establish a hypoxia model; control siRNA transfected group, transfected with control siRNA, and 30 h after the transfection exposed to CoCl(2) for 8 h; and HIF-1 alpha siRNA-transfected group, transfected with HIF-1 alpha siRNA, and 30 h later exposed to CoCl(2) for 8 h. Western blotting was used to detect the protein expression of HIF-1 alpha. Another EC9706 were cultured and divided into 3 groups to be treated as mentioned above, and then exposed to cisplantin or platixal under normoxic or hypoxic condition. 24 hours later 3-(4, 5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay was used to detect the inhibition rates of the cells. Further another EC9706 cells were cultured and then divided into 5 groups: cultured under normoxic condition, cultured under hypoxic condition for 8 h, transfected with control siRNA for 30 h and then under hypoxic condition for 8 h, transfected with HIF-1 alpha siRNA for 30 h and then under hypoxic condition for 8 h. The cell cycle was measured by flow cytometry.
The HIF-1alpha protein expression of the HIF-1alpha siRNA group was significantly lower than those of the untransfected and control siRNA transfected groups. The inhibition rates of the EC9706 cells of the groups treated by cisplatin of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.01). The inhibition rates of the EC9706 cells of the groups treated by platixal of different concentrations under normoxic condition were all significantly higher than the corresponding levels under hypoxic condition (all P < 0.05) Under hypoxic condition, the inhibition rates of the HIF-1alpha siRNA transfected EC9706 cells treated by cisplatin and platixal of different concentrations were all significantly higher than those of the control siRNA transfected and untransfected EC9706 cells (all P < 0.05). Flow cytometry showed that under hypoxic condition the proportion of cells in G(1)-phase of the EC9706 cells was significantly higher, and the proportion of S-phase cells was significantly lower than those of the normoxic group (both P < 0.05), and under the same hypoxic condition the proportion of the EC9706 cells in G(1)-phase was significantly lower, and the proportion the EC9706 cells in S-phase was significantly higher than those of the normoxic group (all P < 0.05).
The cell cycle arrest induced by HIF-1alpha may be the mechanism of the resistance to anticancer drugs of the esophageal squamous cell carcinoma cells under hypoxic condition. Blocking HIF-1alpha in esophageal squamous cell carcinoma cells may reverse the multidrug resistance of the tumor cells, so it may offer an avenue for gene therapy.
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- "Previous studies have shown that siRNA targeting HIF-1α can induce apoptosis of some humam tumor cells and suppress the tumor growth (Chen and Yu, 2009). More recently, a study reported that HIF- 1α siRNA could inhibit the proliferation of ESCC TE-1 cells in vitro (Wu et al., 2007). However, little is known about the effect of HIF-1a siRNA alone or combination with cytotoxic agents on ESCC in vivo. "
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ABSTRACT: The esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances, and a current challenge is the development of effective therapeutic agents. Our present work addressed the effect of HIF-1α siRNA alone or in combination with cisplatin on the growth of ESCC in nude mice.
Xenografts were established by inoculating ESCC TE-1 cells in nude mice, and transplanted tumors were treated with HIF-1α siRNA, cisplatin alone or together. Growth was assessed by measuring tumor volume. HIF-1α mRNA and protein expression were detected using RT-PCR and immunohistochemistry, respectively. Apoptosis of ESCC TE-1 cells was analyzed by flow cytometry.
In our nude mice model, HIF-1α siRNA effectively inhibited the growth of transplanted ESCC, downregulating HIF-1α mRNA and protein expression, and inducing ESCC TE-1 cell apoptosis. Notably when combinated with cisplatin, HIF-1α siRNA showed synergistic interaction in suppressing tumor growth. Furthermore, the proportion of apoptotic cells in HIF- 1α siRNA plus cisplatin group was significantly higher than that in cisplatin or HIF-1α siRNA-treated groups (P<0.05).
Down-regulated HIF-1α expression induced by siRNA could effectively suppress the growth of transplanted ESCC in vivo. HIF-1α siRNA could enhance the cytotoxicity of cisplatin, which suggests that a combination of these two agents may have potential for therapy of advanced ESCC.
Asian Pacific journal of cancer prevention: APJCP 02/2012; 13(2):473-7. DOI:10.7314/APJCP.2012.13.2.473 · 2.51 Impact Factor
Available from: Jianjun Li
- "Hypoxia is well known to induce resistance to drugs and radiation in solid tumors (Piret et al. 2006; Wu et al. 2007) as well as in multicellular tumor spheroids (Wartenberg et al. 2001a, b). The reason that hypoxia contributes to drug resistance in anticancer therapy has not yet been established. "
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ABSTRACT: Hypoxia in tumors is generally associated with chemoresistance and radioresistance. However, the correlation between the heterodimeric hypoxia-inducible factor-1 (HIF-1) and the multidrug resistance (MDR1) gene/transporter P-glycoprotein (P-gp) has not been clearly investigated. This study aims at examining the expression levels of HIF-1α and MDR1/P-gp in human colon carcinoma tissues and cell lines (HCT-116, HT-29, LoVo, and SW480) and ascertaining whether HIF-1α plays an important role in tumor multidrug resistance with MDR1/P-gp.
The expression and distribution of HIF-1α and P-gp proteins were detected in human colon carcinoma tissues and cell lines by immunohistochemistry and immunocytochemistry using streptavidin/peroxidase (SP) and double-label immunofluorescence methods. HIF-1α and MDR1 mRNA expression levels in cell lines were analyzed using RT-PCR under normoxic and hypoxic conditions, respectively.
The immunohistochemical method shows that HIF-1α and P-gp expression were not correlated with gender, age, location, and differentiation degree (P > 0.05). However, the expression of HIF-1α and P-gp at different Dukes' stages and whether involved in lymphatic invasion shows a significant difference (P < 0.05). Correlation analysis displays that HIF-1α protein expression was correlated significantly with P-gp expression (P < 0.01). Double-label immunofluorescence demonstrates that coexpression of HIF-1α and P-gp does exist in human colon carcinoma tissues. The mRNA expression of HIF-1α and MDR1 was detected in the four human colon carcinoma cell lines under both normoxia and hypoxia. Optical density values representing mRNA expression levels of HIF-1α and MDR1 were found to be significantly higher in the same type cells under hypoxic conditions than that under normoxic conditions, respectively (P < 0.01). However, no significant differences of HIF-1α or MDR1 mRNA expression were found among these cell lines, which exposed under the same PaO(2) cultural conditions (P > 0.05). And the immunocytochemistry results were corresponding with the analysis of mRNA expression.
These results suggest that hypoxia induce the expression of HIF-1α and MDR1/P-gp in colon carcinoma and HIF-1α expression may be associated with the gene MDR1 (P-gp) and interactively involved in the occurrence of tumor multidrug resistance.
Journal of Cancer Research and Clinical Oncology 03/2010; 136(11):1697-707. DOI:10.1007/s00432-010-0828-5 · 3.08 Impact Factor
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ABSTRACT: To evaluate the effect of different regimen of radiotherapy on multidrug resistance 1 (MDR1) expression and analyze the role hypoxia-inducible factor 1α (HIF1α) played in the whole process. Fifty-four cell lines established from 96 esophageal cancer biopsy samples were given various doses of fractioned irradiation. The mRNA and protein levels of HIF1α and MDR1 post-irradiation were measured by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. HIF1α-siRNA was used to verify the effect of HIF1α on radiation-mediated MDR1 modulation. In esophageal cancer cells surviving 28 Gy irradiation (2 Gy/f, 14 fractions), MDR1 mRNA expression increased 65.27 ± 5.58%, and HIF1α was elevated by 27.21 ± 2.25%. Interestingly, their expression decreased by 54.38 ± 11.53% and 32.08 ± 4.75% after 7 Gy irradiation (0.5 Gy/f, 14 fractions). HIF1α expression showed a positive correlation with MDR1 expression in the whole process (P < 0.05). Silencing of HIF1α decreased MDR1 expression and blocked changes in MDR1 and HIF1α expression induced by fractioned irradiation. These results indicate that MDR1 is differentially modulated by different doses of fractionated radiation, which should be taken into account when combining radiotherapy and chemotherapy for patients with esophageal cancer.
Diseases of the Esophagus 02/2011; 24(7):481-8. DOI:10.1111/j.1442-2050.2010.01168.x · 1.78 Impact Factor
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