ISI Impact Factor (2004): 1.096

Editor-in-Chief
Prof. Yi-Fei WANG,

Abrogation of heat-shock protein (HSP)70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

Zhi-Gang Zhao¹, Qing-Zheng Ma², Chun-Xiao Xu³

Asian J Androl  2004 Dec; 6: 319-324         

Keywords: prostate carcinoma; heat shock protein (HSP); bcl-2 protein; apoptosis; proliferation

Abstract

Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC-3m cells were treated with 0-16 µmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10 µmol/L antisense HSP70 oligomer for 48 hr or 8 µmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10 µmol/L antisense HSP70 oligomer treatment for 48 hr or 8 µmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abrogates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression, in turn inducing apoptosis and inhibiting cell growth in PC-3m cells.

1 Introduction

Prostate cancer (PCa) is a worldwide significant health care problem due to its high incidence and mortality, in particular, androgen-independent PCa has the worst prognosis. Therefore, it is absolutely necessary to explore a novel modality of treatment for this condition. Heat shock proteins (HSPs) are molecular chaperones that are induced by various environmental and pathophysiological stimuli [1]. Of which the 70-kDa heat shock protein (HSP70) has been shown to play an important role in the regulation of cell growth and transformation [2]. It has been reported that various malignant tumors over-express HSP70, which relates closely to tumorigenesis , malignant development, tumor immunity, resistance to apoptosis and a poor prognosis in the clinical course [2-10]. We have previously reported that human androgen-independent PCa cell line PC-3m has a high expression level of HSP70, which may be involved in the development of androgen-independence in PCa [11]. However, the relationship of HSP70 expression and PC-3m cell growth has not been studied as yet. The present work investigated the effects of abrogating HSP70 expression by using antisense oligonucleotides targeted to human HSP70 mRNA on proliferation of human androgen-independent PCa PC-3m cells. 

2 Materials and methods

The 15-mer nuclease-resistant phosphorothiolate oligodeoxynucleotides (antisense and sense) were synthesized and purified in Shanghai Shenggong Biological engineering corporation (Shanghai, China). HSP70 antisense oligomer (5?CGCGGCTTTGGCCAT-3? was complementary to the initiation codon and 4 downstream codons of human HSP70 mRNA [12]. The corresponding sense oligomer (5?ATGGCCAAAGCCGCG-3? was used as controls. Our previous results on the kinetic studies of HSP70 oligodeoxynucleotide metabolism in PC-3m cells have shown that nuclease-resistant phosphorothiolate oligodeoxynucleotides (antisense and sense) could directly be taken up by PC-3m cells through endocytosis within 90-180 min and exist stably for over 24 hr inside the cells and that these oligomers had a high specificity to bind with the correspondent HSP70 mRNA.

Human androgen-independent PCa cell line PC-3m, kindly gifted from Prof. Li-Zhang Yu (Peking University, Beijing, China), was propagated in RPMI 1640 medium (Hyclone) supplemented with 10 % fetal calf serum (Hyclone) and 100 kU/L penicillin and 100 mg/L streptomycin in a humidified atmosphere of 5 % CO₂ at 37 ℃.

Exponentially growing PC-3m cells at 1×10⁶ cells/mL in culture were treated with HSP70 antisense or sense oligonucleotides at a concentration of 1, 2, 4, 6, 10, 12, 14 and 16 µmol/L for 48 hr or 8 mmol/L for 24, 48, 72, 96 and 100 hr. The culture medium was changed every 24 hr by fresh RPMI 1640 medium, containing the same concentration of HSP70 antisense or sense oligonucleotides . The control cultures were left untreated at 37 ℃ for the same length of time.

The number of viable cells was determined by the Trypan blue dye exclusion test and the percentage inhibition of cell proliferation was calculated by the following formula: Inhibition % =[(N-N_T)/(N-N₀)]×100 %, where N is the number of untreated cells cultured for n days, N0 is the cell number on day 0, and NT is the number of treated cells cultured for n days [13].

PC-3m cells were first treated with 10 µmol/L HSP70 antisense and sense oligomers, respectively, for 24 hr, then harvested and suspended at 5×10⁵ cells/mL in closed Eppendorf tubes (1.5 mL). The tubes were left in a water bath at 42 ℃ for 2 hr, centrifuged at 200 g for 5 min and then the cells were resuspended in fresh medium containing HSP70 antisense and sense oligomers of the same dose before returning to a 37 ℃ incubator for an additional 24 hr. Also, PC-3m cells were treated in parallel with antisense or sense oligomers or heat shock alone.

Apoptotic cells were identified as the percentage by flow cytometry. After treated as described above, cells were harvested and treated with RNase and centrifuged at 200 g for 10 min. The cell pellet was gently resuspended in 1 mL hypotonic fluorochrome solution (PI 50 mg/L, Sigma, in 0.1 % sodium citrate plus 1 mmol/L Tris, 0.1 mmol/L EDTA and 0.1 % Triton X-100) to analyze apoptosis by using a FACScan flow cytometry (Becton Dickinson, USA). Cell-cycle analysis was also performed simultaneously and apoptotic cells appeared in the cell-cycle distribution as cells with DNA content less than that of G₁ cells.

Moreover, DNA fragmentation characteristic of apoptosis was measured using agarose gel electrophoresis . After lysed by lysis buffer containing 50 mmol/L Tris/HCI (pH 8.0), 0.25 % NP40 and 10 mmol/L EDTA, a total of 3×10⁶ PC-3m cells were treated with 20 g/L RNase A (Sigma) at 37 ℃ for 1 hr and subsequently with proteinase K (30 g/L, Sigma) for an additional hour at 37 ℃. The DNA fragments were then precipitated with 2.5 volumes of ethanol and visualized by electrophoresis in 1.5 % agarose gel at 50 V for 2 hr.

Total 2×10⁶ PC-3m cells were lysed in lysis buffer, and then denatured in sample buffer. The protein samples were separated through SDS/PAGE gels, and transferred onto nitrocellulose membranes by electroblotting. Immunoblotting was performed using anti-HSP70 monoclonal antibody (Dako, Glostrup, Denmark) and anti-bcl-2 monoclonal antibody (Dako) for 2 hr, respectively. After washing in 3 % defatted milk, the biotinylated second antibodies (Dako) were incubated for a further hour. The blots were developed using 3, 3'-Diaminobenzidine (Vector Laboratories, USA).

Cell viability was significantly decreased by HSP70 antisense oligomers treatment in a dose- and time-dependent manner, being observed at 1 µmol/L and reaching a maximum at 14 µmol/L for 48 hr or 8 µmol/L for 96 hr (Figure 1, A and B). Moreover, it was found that the antisense treatment not only inhibited cell proliferation but also caused cell death, whereas these effects were not observed in cells treated with sense oligomers.

Figure 1. A, B Dose- (A) and time- (B) dependent inhibition of proliferation by HSP70 antisense oligomers. PC-3m cells treated with various doses of HSP70 antisense or sense oligomers for 48 hr (A), or treated with 8 µmol/L HSP70 antisense or sense oligomers for various lengths of time (B). Results represented as mean±SD of three independent experiments. Student's t-test, P<0.01, cells treated with antisense oligomers vs. sense oligomers.

1.5 % agarose gel electrophoresis showed that treatment of PC-3m cells with 10 µmol/L antisense HSP70 oligomer for 48 hr or 8 µmol/L for 72 hr induced inter-nucleosomal DNA fragmentation in ladder-like patterns (Figure 2, Lane 4 and 5). By contrast, ladder-like patterns of DNA fragments were not found in PC-3m cells treated with sense oligomers of the same doses and incubation time (Figure 2, Lane 3 and 6).

Figure 2. Agarose gel DNA electrophoretic patterns. DNA isolated from antisense or sense oligomers-treated or untreated PC-3m cells. Lane 1: marker; lane 2: untreated; lane 3 and 6: 10 µmol/L sense oligomer treatment for 48 hr and 8 µmol/L for 72 hr, respectively; lane 4 and 5: 10 µmol/L antisense oligomer treatment for 48 hr and 8 µmol/L for 72 hr, respectively.

Kinetic analysis by flow cytometry showed that the apoptotic rate of PC-3m cells induced by HSP70 antisense oligomers was dependent on their doses and incubation intervals, being observed at 1 µmol/L for 48 hr and reaching a maximum at 14 µmol/L for 48 hr or 8 µmol/L for 96 hr. Under the same condition, these effects were not observed in PC-3m cells treated by HSP70 sense oligomers (Figure 3, A and B). Also, sub- G₁ fraction was detected by flow cytometric analysis in the cells treated with HSP70 antisense oligomers, i.e. apoptotic cells appeared in the HSP70 antisense oligomers-treated PC-3m cells. There was a significant decrease in the percentage of G₁ and S-phase fraction among the total PC-3m cells when the number of apoptotic cells increased with the increase in dose and incubation time of HSP70 antisense oligomer (Figure 4).

Figure 3. A, B Kinetics of apoptosis induced by HSP70 antisense oligomers treatment in PC-3m cells. The dose- (A) and time- (B) dependent curves of antisense oligomers-induced apoptosis determined by flow cytometry. PC-3m cells treated with 1-16 µmol/L antisense or sense oligomers for 48 hr (A), or the cells treated with 8 µmol/L HSP70 antisense or sense oligomers for 24 - 100 hr (B). Results are expressed as mean±SD of three independent experiments . Student's t-test, P<0.01, cells treated with antisense oligomers vs. sense oligomers.

Figure 4. Representative DNA fluorescence histograms of fluorescence 2-height (FL-2-H) showing cell cycle distribution and apoptosis percentage. A: untreated; B and C: treated with 10 µmol/L sense HSP70 oligomer for 48 hr and 8 µmol/L for 72 hr, respectively; D-I: treated with 6, 10 and 14 µmol/L antisense HSP70 oligomer for 48 hr and 8 µmol/L for 24, 72 and 96 hr, respectively.

Flow cytometric analysis revealed that the apoptotic rate of PC-3m cells induced by heat shock was significantly higher in the antisense oligomer-treated cells than that in the untreated cells, i.e. treatment with HSP70 antisense oligomers resulted in an increase in the number of apoptotic cells in response to heat shock (Figure 5). By contrast, HSP70 sense oligomers had no effect.

Figure 5. Effects of HSP70 antisense oligomers treatment on apoptosis induced by heat-shock in PC-3m cells. Apoptotic cell numbers calculated by flow cytometry. Results expressed as mean±SD of triplicate samples.

3.5 Abrogation of HSP70 expression by HSP70 antisense oligomer

The results of Western blot showed that the treatment of PC-3m cells with 8 µmol/L HSP70 antisense oligomer for 48 hr and 72 hr or 10 µmol/L for 48 hr resulted in the inhibition of HSP70 expression (Figure 6. Lane D, E and F, respectively), while treatment with 10 µmol/L sense HSP70 oligomer for 48 hr or 8 µmol/L for 72 hr had no effect on HSP70 expression (Figure 6, Lane B and C, respectively). Moreover, it was intriguing that under the same condition, PC-3m cells treated with 10 µmol/L antisense HSP70 oligomer for 48 hr or 8 µmol/L for 72 hr did not show detectable expression of bcl-2 protein (Figure 7, Lane D and E, respectively), whereas those treated with sense HSP70 oligomers showed detectable bcl-2 protein level (Figure 7. Lanes B and C, respectively).

Figure 6. Western blot analysis of HSP70 expression. HSP70 expression level in PC-3m cells untreated (lane A) and treated with 10 µmol/L sense HSP70 oligomer for 48 hr (lane B) or 8 µmol/L for 72 hr (lane C). Inhibition of HSP70 expression in PC-3m cells treated with 8 µmol/L antisense HSP70 oligomer for 48 hr (lane D), 72 hr (lane E) or 10 µmol/L for 48 hr (lane F).

Figure 7. Western blot analysis of bcl-2 protein expression. Bcl-2 expression level in PC-3m cells untreated (lane A) and treated with 10 µmol/L sense HSP70 oligomer for 48 hr (lane B) or 8 µmol/L for 72 hr (lane C). Inhibition of bcl-2 expression in PC-3m cells treated with 10 µmol/L antisense HSP70 oligomer for 48 hr (lane D) or 8 µmol/L for 72 hr (lane E).

4 Discussion

This study has, to our knowledge, first explored the efficacy of antisense HSP70 oligomer for its antiproliferative role against human androgen-independent prostate cancer cell line PC-3m and demonstrated that antisense HSP70 oligomer induced apoptosis and suppressed growth of PC-3m cells in a dose- and time-dependent manner. HSP70 antisense oligomer is found to elevate the number of apoptotic cells and reduce the number of cells at the G₁ and S phase without affecting total cell numbers, suggesting that antisense HSP70 oligomer treatment may cause apoptosis mainly in cells at the G₁ and S phase of the cell cycle, thereby inhibit cell proliferation. Our findings corroborate the previous reports on induction of apoptosis and inhibition of proliferation by abrogation of HSP70 expression in various tumor cells, such as PC-3 and LNCaP [14], Molt-4 tumor cells [15], human oral squamous carcinoma cell HSC-2 [2], monoblastoid U937 and murine fibrosarcoma WEHI-S cells [9] and Jurkat T cells [16]. In addition, the inhibition of HSP70 expression with antisense oligomers can enhance the induction of apoptosis by heat shock. This effect was also observed in Molt-4 cells by Wei et al [15].

Although the mechanism of inducing-apoptosis role of antisense HSP70 oligomer remains obscure, some studies have shown that decreasing HSP70 expression levels in tumor cells could induce apoptosis and inhibit cell growth [2, 15]. Tumor cells having low HSP70 levels have also been shown to respond to the apoptotic stimuli by activation of stress-activated protein kinases, generation of free radicals, early disruption of mitochondrial transmembrane potential, release of cytochrome-c from the mitochondria and the activation of caspase-3-like proteases, suggesting that HSP70 rescues cells from apoptosis later in the death signaling pathway than any other known anti-apoptotic protein [17]. The antisense HSP70 oligomer-treated cells had a consistently decreased expression of bcl-2 protein with inhibited HSP70 expression. A very similar finding was seen in the primary oral SCC cultures and HSC-2 cell line from human oral squamous carcinoma [2]. Although the causes of bcl-2 down-regulation are not exactly clear, abrogation of HSP70 expression by antisense HSP70 oligomers may disrupt HSP70-bcl-2 interactions [2]. Thus, we suggest that HSP70 antisense oligomer can specifically inhibit HSP70 synthesis through sequence-specifical cross-linking with HSP70 mRNA, which may disrupt the interactions of HSP70 and bcl-2 and then result in bcl-2 expression downregulation, thereby activate the signal transduction pathway of apoptosis and lead to apoptosis and cell proliferation inhibition.

In conclusion, antisense HSP70 oligomer can induce apoptosis and inhibit growth in PC-3m cells through abrogating HSP70 expression, which is paralled by bcl-2 downregulation.

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Correspondence to: Dr. Zhi-Gang Zhao, Department of Urology, Shantou University Medical College, Shantou 515031, China.
Tel: +86-754-856 4577
Email: zgzhaodr@yahoo.com.cn
Received 2003-03-10   Accepted 2004-07-22