| Background and objectivesColon cancer is one of the most common malignant tumors and chemotherapy plays an important role in its treatment. However, sensitivity to different chemotherapeutic schedules among different individual patients shows very various. Many cancer patients have poor treatment outcome. Multidrug resistance (MDR) to antineoplastic drugs is one of the major reasons why chemotherapy-based treatment appears failure. So-called MDR is defined as tumor cells resist to not only previously used cytostatic agents, but also cross-resist to other drugs with different structure and function. In the multitudinous mechanisms of MDR, the high expression of human MDR1 gene and P-glycoprotein (P-gp) which is encoded by MDR1 is the relative focus in its research. P-gp is a kind of transmembrane drug transporter depending on ATP activity and capable of transporting a variety of structurally and functionally diverse chemotherapy drugs. It can transport the drugs out of cytomembrane and maintain a low concetration of chemotherapeutics in the inner of tumor cells. The tumor cells with overexpression of P-gp usually show a resistant to many chemotherapeutics which leads to the failure of chemotherapy.Hypoxia is a common feature of many malignant tumors including colon carcinoma. The physiology and biochemistry of tumor cells changes to adapt hypoxia. Hypoxia- inducible factor-1 (HIF-1) is a key factor in altering the biological characteristics of tumors. It has been reported that HIF-1αprotein was overexpressed in multiple types of human cancers and it was associated with the prognosis of many cancers. More importantly, many studies indicated that hypoxia helped improve chemotherapy and radiotherapy resistance of tumor [1]. The reason that hypoxia contributes to drug resistance in anticancer therapy has not yet been established. It is unclear yet whether and how HIF-1αinvolves in the MDR occurrence of colon carcinoma with the interaction of MDR1/P-gp. Could it be possible that the reversal of colon carcinoma MDR was achieved at the study level of gene if HIF-1αexpression was specifically inhibited? Better understanding the influence of colon carcinoma MDR by hypoxia will help improve the effect of chemotherapy.In the present study, the expression and distribution of HIF-1αand P-gp protein were examined in human colon carcinoma tissue. Also, the expression level of HIF-1αand MDR1/P-gp were detected in four types of colon carcinoma cell lines respectively under normoxic and hypoxic conditions. Lentiviral vector mediated microRNA (miRNA) RNAi system of target gene HIF-1αand MDR1 was respectively constructed. Subsequently, after hypoxia induced MDR phenotype of LoVo cell line was established depending on multicellular spheroids (MCS) cell culture system and the expression of HIF-1αand MDR1 were efficiently silenced, the sensitivity to chemotherapeutics of LoVo MCS and the drugs induced apoptosis level were examined for observing the reversal efficiency of MDR. All of above is to explore the probable molecular mechanisms and feasibility of reversing colon carcinoma MDR by focused on the target gene HIF-1αand expects to provide a theory basis and experimental evidence for later relative applications in clinical treatment.Methods1. To investigate the expression and significance of hypoxia- inducible factor-1α(HIF-1α) and P-gp in human colon carcinoma tissue and the relationship with the certain clinicopathological features, such as tumor location, differentiation degrees, Dukes'stages and lymphatic invasion, the expression and distribution of HIF-1αand P-gp proteins were detected in human colon carcinoma tissue by immunohistochemistry using streptavidin/ peroxidase (SP) and double-label immunofluorescence methods.2. To explore the influence and relationship between hypoxic microenvironment in tumor and the expression levels of HIF-1αand MDR1/P-gp, the mRNA and protein expression levels of HIF-1αand MDR1 were analyzed by using RT-PCR and immunocytochemistry methods under normoxic and hypoxic conditions respectively in four types of human colon carcinoma cell lines.3. Pre-miRNA RNAi sequences for target gene HIF-1αand MDR1 were designed and synthesized respectively. After the two specific miRNA RNAi expression vectors with report gene EmGFP respectively targeting the gene of human HIF-1αand MDR1 were constructed and identified by restriction enzyme digestion and sequencing, the RNAi plasmids of HIF-1αand MDR1 were respectively transfected into LoVo cells by using liposome LipofectamineTM 2000. Stable positive clone was selected by using blasticidin. The suppression effects targeting HIF-1αand MDR1 were identified by semi-quantification RT-PCR. The RNAi plasmids with better suppression effects were selected for subsequent experiments on constructing miRNA lentivirus expression clones.4. Two miRNA lentivirus expression colons (pLenti6/V5-GW/EmGFP-miR-HIF-1α& pLenti6/V5-GW/EmGFP-miR-MDR1) were constructed by using Gateway recombination technics and co-transfected into 293FT cells with the ViraPower? packaging mix. The titer was examined after infecting NIH/3T3 cells with virus supernate. Then LoVo cells were infected with the two lentiviral vector mediated miRNA RNAi systems and stably selected by blasticidin. Subsequently, the mRNA and protein expression levels of HIF-1αand MDR1 were detected respectively by semi-quantification RT-PCR and Western blot and the relationship between HIF-1αand MDR1 was further analyzed at the level of proteins.5. Parental LoVo MCS and stable HIF-1α(-) or MDR1(-) were respectively established by three dimensional cell culture method. The LoVo MCS with MDR phenotype was further achieved by hypoxia induction and cell cycle distribution of each LoVo MCS group was examined by flow cytometry (FCM). Thereafter, the suppression effects of both LVV-miRNA RNAi systems were investigated by Western blot at the level of LoVo MCS.6. The chemotherapy sensitivity changes to LoVo MCS of Adriamycin (ADR), Vincristine (VCR), 5-Fluorouracil (5-Fu) and Irinotecan (CPT-11) were examined by MTT under normoxia and hypoxia respectively. Also, the chemotherapy sensitivity changes to LoVo MCS with stable HIF-1α(-) or MDR1(-) were detected and the corresponding reversal of MDR was observed.7. Apoptosis level induced by the above drugs of each LoVo MCS group was respectively examined by FCM.Results1. HIF-1αprotein expression was predominantly localized in cytoplasm of tumor cells, part of in nuclei, especially in the margin of tumor necrotic and infiltrating regions. P-gp expression was mainly localized in cytoplasm and cytomembrane of tumor cells. According to the 58 tissue specimens, the statistical analysis of HIF-1αand P-gp expression was as follows. The rates of positive HIF-1αand P-gp protein expression were 58.6% (34/58) and 46.6% (27/58) respectively. HIF-1αand P-gp expression were not correlated with gender, age, location and differentiation degree (P>0.05). The rates of positive HIF-1αand P-gp expression were 74.1% (20/27) and 62.3% (17/27) respectively at Dukes'stages C and D, which were significantly higher than that of 45.2% (14/31) and 32.3% (10/31) at Dukes'stages A and B (P<0.05). Furthermore, the rates of positive HIF-1αand P-gp expression were both 78.9% (15/19) in those involved in lymphatic invasion, which were significantly higher than that of 48.7% (19/39) and 30.8% (12/39) in those without lymphatic invasion (P<0.05). The Spearman analysis showed that the expression level of HIF-1αwas significantly associated with P-gp expression (r=0.574, P<0.01). Double-label immunofluorescence demonstrates that coexpression of HIF-1αand P-gp does exist in human colon carcinoma tissue.2. The mRNA expression of HIF-1αand MDR1 were detected in four types of human colon carcinoma cell lines (HCT-116, HT-29, LoVo and SW480) very weakly in normoxia and strongly in hypoxia. Optical density values representing mRNA expression levels of HIF-1αand MDR1 were found to be significantly higher in the same type cells under hypoxic cultural conditions for 24 h than that under normoxic conditions respectively (P<0.01). However, no significant differences of HIF-1αor MDR1 mRNA expression were found among each cell line under the same PaO2 cultural conditions (P>0.05). And the immunocytochemistry results were corresponding with the analysis of mRNA expression.3. Two specific miRNA RNAi expression vectors (pcDNA?6.2-GW/EmGFP-miR- HIF-1α(MDR1)) with report gene EmGFP were successfully constructed and transfected into LoVo cells. The semi-quantification RT-PCR results showed that compared with non-treated and neg-control group, the mRNA expression of target gene could specifically and efficiently inhibited by HIF-1αor MDR1 miRNA RNAi plasmid (P<0.01). Meanwhile, MDR1 mRNA expression level in HIF-1αmiRNA RNAi group was significantly lower than that of non-treated and neg-control group (P<0.01). Conversely, HIF-1αmRNA expression level in MDR1 miRNA RNAi group was similar to that of non-treated and neg-control group (P>0.05). The RNAi plasmids with better suppression effects of HIF-1αand MDR1 were selected for the next experiments.4. Two miRNA lentivirus expression colons of target gene HIF-1αand MDR1 (pLenti6/V5-GW/EmGFP-miR-HIF-1α& pLenti6/V5-GW/EmGFP-miR-MDR1) were successfully constructed. After stably infecting LoVo cells, the data of semi-quantification RT-PCR and Western blot demonstrated that the mRNA and protein expression levels of HIF-1αand MDR1 in LVV-HIF-1αmiR group were significantly lower than that of non-treated and neg-control group (P<0.01). However, in LVV-MDR1 miR group, only the mRNA and protein expression levels of gene MDR1 were statistically lower than that of non-treated and neg-control group (P<0.01), while there were no differences of HIF-1αmRNA and protein expression levels between each group (P>0.05). This was consistent with the results of using HIF-1αand MDR1 RNAi plasmids.5. Parental LoVo MCS model and stable HIF-1α(-) or MDR1(-) were successfully established. The results of cell cycle by FCM showed that compared with the parental LoVo MCS, the G1-phase ratio of LoVo MCS in hypoxia group significantly increased (P<0.01), and the ratio of S-phase and G2-phase decreased (P<0.01). In addition, compared with parental LoVo MCS under hypoxic conditions, the G1-phase ratio of LoVo MCS/LVV-HIF-1αmiR group significantly decreased (P<0.01) accompanying with S-phase and G2-phase ratio rose (P<0.01). However, there was no significant differences of each phase between LoVo MCS/LVV-MDR1 miR and parental LoVo MCS group under hypoxia (P>0.05). Western blot showed that both HIF-1αand MDR1 miR lentivirus RNAi systems specifically and efficiently worked at the level of LoVo MCS (P<0.01), and the inhibitation ratio of target gene HIF-1αand MDR1 was respectively 76.0% and 74.5%.6. The chemotherapy sensitivities to LoVo MCS of the four drugs were all decreased at different extent after handling with hypoxia. The values of fifty percent inhibiting concentration (IC50) in the hypoxia group were all higher than that of normoxia group (ADR, VCR and 5-Fu, P<0.01; CPT-11, P>0.05). After silencing gene HIF-1α, the sensitivities to ADR, VCR and 5-Fu of LoVo were enhanced at different extent. The decreased extents of each drug resistance and relative reversed ratio show as follows: ADR, from 9.92-fold to 2.62-fold, 81.78% (P<0.01); VCR, from 6.10-fold to 1.92-fold, 82.04% (P<0.01); 5-Fu, from 4.94-fold to 2.26-fold, 68.00% (P<0.01); and CPT-11, with no statistical difference, from 1.18-fold to 1.08-fold (P>0.05). Likewise, the same types of parameter and data after silencing gene MDR1 show as follows: ADR, from 9.92-fold to 3.21-fold, 5.24% (P<0.01); VCR, from 6.10-fold to 1.71-fold, 86.04% (P<0.01). Notably, the IC50 values of 5-Fu and CPT-11 were similar to that of parental LoVo MCS group, with no statistical difference (P>0.05), which suggested that there was no significant influence on chemotherapy sensitivities of them after silencing gene MDR1.7. The data of apoptosis by FCM demonstrated that compared with parental LoVo MCS under normoxic cultrual conditions, the apoptosis levels by each drug induced were all decreased at various extents in hypoxia group. The proportion to parental LoVo MCS in normoxia group of apoptosis ratio induced by ADR, VCR and 5-Fu was 11.10%, 12.30% and 30.58% respectively with a statistical significance (P<0.01). However, the induced apoptosis ratio of CPT-11 was similar to normoxia group (P>0.05), though with a descent. After efficiently silencing the target gene HIF-1αor MDR1, the changes of drug induced apoptosis levels showed as follows. After efficiently silencing the target gene HIF-1α, the induced apoptosis level by ADR, VCR and 5-Fu was remarkably increased (P<0.01) and was 9.18-fold, 7.38-fold, and 2.64-fold respectively contrast to that of LoVo MCS with MDR phenotype. Nevertheless, the apoptosis level induced by CPT-11 was only 1.05-fold to hypoxia group, with no statistical significance (P>0.05). As far as the LoVo MCS/LVV-MDR1 miR group was concerned, the apoptosis level induced by ADR and VCR was notably up-regulated (P<0.01) and was 8.69-fold, 7.23-fold respectively contrast to that of LoVo MCS with MDR phenotype. However, there was no significant difference of the apoptosis level induced by 5-Fu and CPT-11 between LoVo MCS/LVV-MDR1 miR group and LoVo MCS with MDR phenotype (P>0.05).Conclusions1. Overexpression and coexpression of HIF-1αand P-gp protein do exist in human colon carcinoma tissue. The expression of HIF-1αand P-gp is not correlated with gender, age, location, and differentiation degree. But the expression of HIF-1αand P-gp at different Dukes'stages and whether involved in lymphatic invasion shows a significant difference.Association analysis displays that the expression of HIF-1αprotein is correlated significantly with P-gp.2. The mRNA and protein expression levels of HIF-1αand MDR1 in colon carcinoma cells can be remarkably up-regulated by hypoxia induction. HIF-1αmay participate in the occurrence of tumor MDR by the direct or indirect interaction with MDR1 (P-gp).3. The target gene HIF-1αand MDR1 can specifically and efficiently silenced in LoVo cells both at monolayer and MCS levels by the two lentiviral vector mediated miRNA RNAi systems. Furthermore, MDR1/P-gp expression can be simultaneously, pronounced down-regulated by silencing the expression of HIF-1α. It suggests that HIF-1αmay probably directly or indirectly activate or induce the up-regulation of MDR1 expression as an upstream signaling molecule under hypoxic conditions.4. Hypoxia can induce parental LoVo MCS generating MDR phenotype and turning into an effective drug resistance model to ADR, VCR and 5-Fu which is also less sensitivity to CPT-11, but with no statistical significance. Silencing the target gene MDR1 can reverse the drug resistance to ADR and VCR via the classical pathway mediated by P-gp in LoVo MCS. Silencing the target gene HIF-1αcan efficiently reverse the drug resistance to ADR, VCR and 5-Fu in LoVo MCS. As for HIF-1α, the mechanism of reversing MDR may be correlated with many factors, such as inducing the expression of MDR1/P-gp, involving in the regulation of cell cycle, inhibiting apoptosis, etc.5. Silencing the target gene HIF-1αcan apparently promote the cells arrested in G1-phase transiting into S-phase. The combination use of LVV-HIF-1αmiR RNAi system and ADR, VCR or 5-Fu can significantly induce apoptosis of LoVo MCS with MDR phenotype and consequently improve the chemotherapy effects.6. The chemotherapy resistance mechanism of CPT-11 is independent on the classical drug resistance pathway mediated by P-gp. It may count on the inhibition of HIF-1αby its active metabolic production SN-38 or other pathway to keep its sensitivity to a certain extent in the hypoxic microenvironment of tumor. |
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