Hydroxychloroquine facilitates autophagosome formation but not degradation to suppress the proliferation of cervical cancer SiHa cells

Abstract

Hydroxychloroquine (HCQ), the hydroxylated analog of chloroquine, is an antimalarial lysomotropic agent that inhibits autophagy due to lysosomal acidification, and subsequently blocks the fusion of autophagosomes with lysosomes which leads to the accumulation of autophagosomes that may accelerate tumor cell death. Given these hypothesis the aim of this study was to investigate the effects of HCQ in the inhibition of autophagy and the induction of apoptosis in cervical cancer SiHa cells. Cervical cancer SiHa cells were cultured with Hank's balanced salt solution (HBSS) as positive control of autophagy or treated with HCQ as part of the experimental groups. LC3 and P62/SQSTM1 were detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively in order to evaluate initially autophagosome formation and their degradation. Specific green fluorescent protein (GFP)-LC3 was subsequently detected by fluorescence microscopy in order to confirm the formation of autophagosomes. MTT and flow cytometry were adopted respectively to assess the proliferation and apoptosis of the SiHa cells. miRNA-9* was also investigated. The results demonstrated that HCQ increased the expressions of LC3 mRNA and LC3II protein and GFP-LC3 signalling but reduced the expression of p62/STSQM1 in cervical cancer SiHa cells. These results indicated HCQ has the ability to inhibit autophagy as incapable of degrading the autophagosome. However, HCQ may promote SiHa cell apoptosis as the MTT, apoptotic assay and miRNA-9* results revealed. HCQ has the ability to inhibit autophagy by blocking the degradation of autophagosomes and subsequently facilitates the apoptosis of cervical cancer SiHa cells.

Keywords: apoptosis; autophagy; cervical cancer; hydroxychloroquine.

Figure 1

Expression of LC3 mRNA in cervical cancer SiHa cells treated with HBSS or HCQ. SiHa cells were treated with 20 μmol/l HCQ or starvation for 1, 6, 12 and 24 h. The relative expression of LC3 mRNA was detected by real-time polymerase chain reaction. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05 vs. full medium control. HBSS, Hank’s balanced salt solution; HCQ, hydroxychloroquine.

Figure 2

Expression of LC3 protein in cervical cancer SiHa cells treated with HBSS. Western blot analysis for the expression of LC3 protein in SiHa cells starved for 1, 6, 12 and 24 h. HBSS, Hank’s balanced salt solution.

Figure 3

Expression of LC3 protein in cervical cancer SiHa cells treated with HCQ. Western blot analysis for the expression of LC3 protein in SiHa cells treated with 20 μmol/l HCQ for 1, 6, 12 and 24 h. HCQ, hydroxychloroquine.

Figure 4

Specific GFP-LC3 combined fluorescence microscopy in cervical cancer SiHa cells treated with HBSS or HCQ. Cells were treated with 20 μmol/l HCQ and starvation for 12 h and stained with specific green fluorescent protein-LC3. Cell morphology was observed by fluorescence microscopy (red arrows indicate autophagosomes or LC3 accumulation). FBS, fetal bovine serum; HCQ, hydroxychloroquine; HBSS, Hank’s balanced salt solution.

Figure 5

Relative expression of P62 mRNA in SiHa cells. SiHa cells were treated with 20 μmol/l HCQ and starvation for 1, 6, 12 and 24 h. The relative expression of LC3 mRNA was detected by real-time polymerase chain reaction. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05 vs. full medium control. HBSS, Hank’s balanced salt solution; HCQ, hydroxychloroquine.

Figure 6

Relative expression of protein in SiHa cells. SiHa cells were treated with 20 μmol/l HCQ and starvation for 1, 6, 12 and 24 h. Western blot analysis was used to determine the LC3 protein expression in SiHa cells. HCQ, hydroxychloroquine; HBSS, Hank’s balanced salt solution.

Figure 7

HCQ inhibits the proliferation of cervical cancer SiHa cells. SiHa cells were treated with 20 μmol/l HCQ or starvation for 1, 6, 12 and 24 h. Cell viability was determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05 vs.. full medium control. HCQ, hydroxychloroquine.

Figure 8

Flow cytometry detection of apoptotic and dead cells induced by HCQ or HBSS. SiHa cells were treated with 20 μmol/l HCQ and starvation for 1, 6, 12 and 24 h. HCQ, hydroxychloroquine; HBSS, Hank’s balanced salt solution.

Figure 9

Quantitation of apoptotic cell ratio. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05 vs. full medium control. HCQ, hydroxychloroquine; HBSS, Hank’s balanced salt solution.

Figure 10

Relative expression of miRNA-9^* in cervical cancer SiHa cells treated with HBSS or HCQ. miRNA-9^* levels detected by stem-loop reverse transcription PCR with SYBR Green PCR master mix in SiHa cells treated with 20 μmol/l HCQ and starvation for 1 and 12 h. Data are presented as the mean ± standard deviation (n=3). ^*P<0.05 vs. full medium control. HCQ, hydroxychloroquine; HBSS, Hank’s balanced salt solution; PCR, polymerase chain reaction.