| OBJECTIVE:To observe the multidrug resistance reversal effect and mechanism of GXHSWAQ-1 in cisplatinum-resistant ovarian cancer cells SKOV3/DDP (short for cisplatin) in vitro.METHODS : We established the cisplatin-resistant ovarian cancer cells SKOV3/DDP by treating the SKOV3 parental ovarian cancer cells continuously with low concentration of DDP. Methyl thiazolyl tetrazolium (MTT) method was used to detect the multidrug resistance of SKOV3/DDP to multianticarcinogen and the reversal effect of GXHSWAQ-1. The influence of GXHSWAQ-1 on cell cycle,apoptosis and ROS was determined by flow cytometry. The expression of HIF-1α, STAT1 , GSTP1 and other resistance-associated genes was detected by Real time PCR.The expression of HIF-1αprotein was studied by Western blotting assay.RESULTS:1. Detection of resistant factor:The half maximal inhibitory concentration (IC50) of SKOV3 and SKOV3/DDP were 8.6 mg/L and 24.2 mg/L to DDP,63.3 mg/L and 266.7 mg/L to CBP (short for carboplatin). The resistant factors (RF) of SKOV3/DDP to DDP and CBP were 2.8 and 4.2 respectively.2. Influence of the survival rate of GXHSWAQ-1 on SKOV3 and SKOV3/DDP : Though the inhibition of GXHSWAQ-1 on SKOV3 and SKOV3/DDP both elevated when the concentration increased. The inhibitory rate of SKOV3 and SKOV3/DDP were both lower than 10% when the concentration of GXHSWAQ-1 was less than 7.8mg/L (including 7.8mg/L).3. Reversal effect of GXHSWAQ-1: Noncytotoxic dose of GXHSWAQ-1 reversed the cisplatin-resistance of SKOV-3/DDP,the reversal index (RI) was 1.91 and 1.30 respectively for 7.8 mg/L and 3.9 mg/L GXHSWAQ-1. Noncytotoxic dose of GXHSWAQ-1 reversed the carboplatin-resistance of SKOV-3/DDP,the reversal index (RI) was 2.59 and 1.84 respectively for 7.8 mg/L and 3.9 mg/L GXHSWAQ-1.4. Study of apoptosis, cell cycle and reactive oxygen species (ROS) in SKOV3/DDP: Compared to SKOV3/DDP treated with DDP or CBP alone, the apoptosis rate of SKOV3/DDP was significantly raised (P<0.01) when 7.8 mg/L GXHSWAQ-1 was combined. The decrease of G0-G1 phase (P<0.01) cell populations and increase of S and G2-M phase (P<0.01) cells were observed. GXHSWAQ-1 can induce SKOV3/DDP ROS to increase in a dose-dependent manner. The ROS of SKOV3/DDP was significantly raised when 7.8 mg/L GXHSWAQ-1 was combined with DDP or CBP (P<0.01).5. Expression of HIF-1α,STAT1,GSTP1 mRNA: Compared to SKOV3, expression of HIF-1α,STAT1,GSTP1 mRNA in SKOV3/DDP was significantly raised(P<0.01). 3.9mg/L and 7.8mg/L GXHSWAQ-1 both downregulated HIF-1α,STAT1 mRNA expression in a dose-dependent manner. Though 7.8mg/L GXHSWAQ-1 downregulated GSTP1 mRNA expression,3.9mg/L GXHSWAQ-1 had no such effects. The expression of HIF-1α,STAT1,GSTP1 mRNA was significantly downregulated when 7.8 mg/L GXHSWAQ-1 was combined with DDP or CBP (P<0.01).6. Expression of HIF-1αprotein: Compared to SKOV3, expression of HIF-1αprotein in SKOV3/DDP significantly raised (P<0.01). There were no significant changes in HIF-1αprotein expression when DDP or CBP were used alone. 3.9mg/L,7.8mg/L and 15.6mg/L GXHSWAQ-1 downregulated HIF-1αprotein expression in a dose-dependent manner. The expression of HIF-1αprotein was significantly downregulated when 7.8 mg/L GXHSWAQ-1 was combined with DDP or CBP (P<0.01).CONCLUSION:1. We successful establised the cisplatin-resistant ovarian cancer cell line SKOV3/DDP.2. GXHSWAQ-1 can partly reverse the cisplatin or carboplatin resistant ovarian cancer cells. The reversal effect is expressed in a dose-dependent manner. 3. GXHSWAQ-1 can induce cells apoptosis by increasing ROS generation or cell cycle redistribution. It is thought that the action may be related to HIF-1αand GSTP1 downregualtion, also maybe correlated with STAT1 signal pathway inhibition.
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