α-lipoic acid modulates prostate cancer cell growth and bone cell differentiation

Abstract

Prostate cancer (PCa) progression leads to bone modulation in approximately 70% of affected men. A nutraceutical, namely, α-lipoic acid (α-LA), is known for its potent anti-cancer properties towards various cancers and has been implicated in treating and promoting bone health. Our study aimed to explore the molecular mechanism behind the role of α-LA as therapeutics in preventing PCa and its associated bone modulation. Notably, α-LA treatment significantly reduced the cell viability, migration, and invasion of PCa cell lines in a dose-dependent manner. In addition, α-LA supplementation dramatically increased reactive oxygen species (ROS) levels and HIF-1α expression, which started the downstream molecular cascade and activated JNK/caspase-3 signaling pathway. Flow cytometry data revealed the arrest of the cell cycle in the S-phase, which has led to apoptosis of PCa cells. Furthermore, the results of ALP (Alkaline phosphatase) and TRAP (tartrate-resistant acid phosphatase) staining signifies that α-LA supplementation diminished the PCa-mediated differentiation of osteoblasts and osteoclasts, respectively, in the MC3T3-E1 and bone marrow macrophages (BMMs) cells. In summary, α-LA supplementation enhanced cellular apoptosis via increased ROS levels, HIF-1α expression, and JNK/caspase-3 signaling pathway in advanced human PCa cell lines. Also, the treatment of α-LA improved bone health by reducing PCa-mediated bone cell modulation.

Keywords: Bone modulation; HIF-1α; Osteoblasts; Osteoclasts; Prostate cancer; Reactive oxygen species (ROS); p-JNK; α-LA.

Figure 1

Effect of increasing concentrations of α-LA on PCa cell viability determined by MTT assay (A). Cells were treated with various concentrations of α-LA for 48 h, and loss of viability was measured by MTT assay. Values are expressed as mean ± SEM. The cell cycles of 22Rv1 and C4-2B cells were treated with 500 µM of α-LA. (B) DNA content in different cell cycle phases was assessed by propidium iodide staining of control and treated cells, followed by FACS analysis. Cell cycle analysis showed that after α-LA treatment, cells were arrested at the S phase. (C) Quantitative analysis of DNA content was shown as mean ± SEM (n = 3). (D) Western blot analysis of 22Rv1 and C4-2B performed after α-LA treatment reveals that the cell cycle arrest facilitates via upregulation of p21 protein and downregulation of cyclin E and pRb protein. Statistical significance was calculated using two-way ANOVA. ****p < 0.0001; ***p < 0.0002; **p < 0.001; *p < 0.05.

Figure 2

Effect of α-LA on apoptotic assay in PCa cell lines. (A). The percentage of viable and apoptotic cells analysis with respect to control (untreated) in 22Rv1 and C4-2B cells treated with 500 µM of α-LA for 48 h as determined by a flow cytometer of annexin-V Cy-5/PI- dual stained cells. (B) Quantitative analysis of these micrographs was shown as mean ± SEM. (C) Representative western blots of pro and anti-apoptotic genes in 22Rv1 and C4-2B cells after the treatment as described. Statistical significance was calculated using a t-test. *p < 0.05; **p < 0.001.

Figure 3

α-LA induced-ROS generation in PCa cell lines. (A) α-LA induced ROS generation evaluated fluorescence microscope with DCFH-DA. Scale bars = 200 µm. (B) ROS levels induced by α-LA were measured using a fluorescent plate reader, analyzed statistically, and presented as a bar graph as mean ± SEM. (C) Western blots of ROS scavenging proteins after treatment of α-LA in 22Rv1 and C4-2B as described. (D) Effect of pre-treatment with NAC on the α-LA-induced cell death. 22Rv1 and C4-2B cells were preincubated with 0.1 mM of NAC and then treated with 2000 µM of α-LA for 48 h. Statistical significance was calculated using a t-test. P-value ****p < 0.0001 (compared to control) or two-way ANOVA. ^####p < 0.0001 (compared to 2000 µM of α-LA without NAC).

Figure 4

Western blot analysis showing expression of HIF-1α, pJNK, and cleaved caspase-3. The blotting depicted a higher expression of HIF-1α, pJNK, and cleaved caspase-3 upon α-LA treatment assessed in whole cell lysates isolated from control and α-LA treated 22Rv1, and C4-2B. β-actin was used as the loading control.

Figure 5

α-LA inhibited the invasion and cell migration potential of PCa cells. (A,B) Wound assay of 22Rv1 cells treated with 500 µM of α-LA for 48 h. Imaging has been done at a magnification of 10x (t = 0 h, t = 24 h & 48 h) and analyzed through Image J software. Scale bars = 400 µm. (C,D) Migration assay of 22Rv1 and C4-2B cells treated with 500 µM of α-LA for 48 h. Migrated cells were stained with crystal violet, and imaging was done in five different areas at a magnification of 10x. Stained migrated cells were counted and analyzed statistically by using a t-test. Scale bars = 400 μm. (E,F) Representative images of colony formation assay in 22Rv1 and C4-2B cells after 14–20 days of treatment with 500 µM of α-LA. Quantification was done by counting the number of colonies and represented as a bar diagram. (G) Western blot analysis of EMT markers after the treatment as described. Statistical significance was calculated using a t-test, and results are expressed as mean ± SEM. *p < 0.05, ***p < 0.005, ****p < 0.0001.

Figure 6

α-LA treatment inhibits PCa-mediated osteoblast differentiation. (A) PCa interaction with bone cells promotes osteoblast differentiation, which was inhibited by treatment with 100 nM of α-LA. Undifferentiated MC3T3-E1 cells were treated with various proportions of 22Rv1 condition medium (20%, 40%, 50%, and 50% with 100 nM of α-LA) for 48 h and subjected to alkaline phosphatase activity. (B) Representative ALP staining of MC3T3-E1 cells after 10 days of treatment with the alone 50% condition medium of 22Rv1 cells and 50%CM with 100 nM of α-LA. (C) MC3T3-E1 cells were treated with the control, 20%, 50% CM, and 50% CM with 100 nM α-LA for 48 h in the differentiation medium of osteoblast. Total RNA was harvested, RT–qPCR for osteogenic genes (alkaline phosphatase: ALP, Runx-2, Col-1a, and osteocalcin) was performed, and gene expression was normalized to that of β-actin. Statistical significance was calculated using one-way ANOVA or t-test, and results were presented as mean ± SEM, p-value; **** p < 0.0001; **p < 0.0080, *p < 0.05 (compared to control); ^####p < 0.0001; ^###p < 0.0003 (compared to 50%CM without α-LA).

Figure 7

α-LA treatment inhibits PCa-mediated osteoclastic differentiation and function. (A) PCa interaction with bone cells promotes osteoclast differentiation, which was inhibited by the treatment with 100 nM of α-LA. BMMs from C57BL/6J mice were cultured for 7 days with M-CSF, RANKL, and various proportions of 22Rv1 condition medium (20%, 50% and 50% with 100 nM of α-LA). Total RNA was harvested, RT–qPCR for osteoclast markers (TRAP, NFATc1, carbonic anhydrase, c-fos, and cathepsin K) was performed, and gene expression was normalized to that of β-actin. (B) Representative TRAP staining of BMMs cells after 7 days of treatment with alone 50% condition medium of 22Rv1 cells and 50%CM with 100 nM of α-LA. (C) The number of TRAP + ve cells was counted and represented as a bar diagram. (D) Representative images of pit resorption assay of BMMs cells after 7 days of treatment as described. Statistical significance was calculated using one-way ANOVA or t-test, and results were presented as mean ± SEM, ****p < 0.0001; ***p < 0.0004; **p < 0.008; *p < 0.05 (compared to control); ^####p < 0.0001; ^###p < 0.0003; ^##p < 0.003; ^#p < 0.01 (compared to 50%CM without α-LA).