Augmentation of Docetaxel-Induced Cytotoxicity in Human PC-3 Androgen-Independent Prostate Cancer Cells by Combination With Four Natural Apoptosis-Inducing Anticancer Compounds

Abstract

Docetaxel (DTX) is the treatment of choice for metastatic castration-resistant prostate cancer. However, developing drug resistance is a significant challenge for achieving effective therapy. This study evaluated the anticancer and synergistic effects on DTX of four natural compounds (calebin A, 3^'-hydroxypterostilbene, hispolon, and tetrahydrocurcumin) using PC-3 androgen-resistant human prostate cancer cells. We utilized the CellTiter-Glo^® luminescent cell viability assay and human PC-3 androgen-independent prostate cancer cells to determine the antiproliferative effects of the four compounds alone and combined with DTX. Cytotoxicity to normal human prostate epithelial cells was tested in parallel using normal immortalized human prostate epithelial cells (RWPE-1). We used cell imaging and quantitative caspase-3 activity to determine whether these compounds induce apoptosis. We also measured the capacity of each drug to inhibit TNF-α-induced NF-kB using a colorimetric assay. Our results showed that all four natural compounds significantly augmented the toxicity of DTX to androgen-resistant PC-3 prostate cancer cells at IC₅₀. Interestingly, when used alone, each of the four compounds had a higher cytotoxic activity to PC-3 than DTX. Mechanistically, these compounds induced apoptosis, which we confirmed by cell imaging and caspase-3 colorimetric assays. Further, when used either alone or combined with DTX, the four test compounds inhibited TNF-α-induced NF-kB production. More significantly, the cytotoxic effects on normal immortalized human prostate epithelial cells were minimal and non-significant, suggesting prostate cancer-specific effects. In conclusion, the combination of DTX with the four test compounds could effectively enhance the anti-prostate cancer activity of DTX. This combination has the added value of reducing the DTX effective concentration. We surmise that calebin A, 3^'-hydroxypterostilbene, hispolon, and tetrahydrocurcumin were all excellent drug candidates that produced significant antiproliferative activity when used alone and synergistically enhanced the anticancer effect of DTX. Further in vivo studies using animal models of prostate cancer are needed to confirm our in vitro findings.

Keywords: 3′-hydroxypterostilbene; calebin A; docetaxel; hispolon; polyphenolic compounds; tetrahydrocurcumin.

Figure 1.

(A) CBA at 40 μM IC50 concentration induced significant PC-3 cell death when used alone and augmented docetaxel (DTX) cytotoxicity compared to control untreated cells and PC-3 cells treated with DTX alone (112 nM). (B) No significant cell death was detected in RWPE-1 immortalized normal human prostate epithelial cells. Data is an average of three experimental repeats.

Figure 2.

(A) 3^′-Hydroxypterostilbene (HPSB) at 40 μM IC50 induced a significant reduction of cell viability in human androgen resistance prostate PC-3 cancer cells and augmented docetaxel (DTX) anti-prostatic activity compared to untreated control and to PC-3 treated with DTX alone (112 nM). (B) No significant reduction in cell viability of RWPE-1 immortalized normal human prostate epithelial cells. Control cells were treated with vehicles. Data is an average of three experimental repeats.

Figure 3.

(A) Tetrahydrocurcumin (THC) at 40 μM IC50 concentration induced a significant reduction of cell viability in human androgen resistance prostate PC-3 cancer cells and augmented docetaxel (DTX) anti-prostatic activity when compared to untreated control and to PC-3 cell treated with DTX alone (112 nM). (B) No significant reduction in cell viability of RWPE-1 immortalized normal human prostate cells. Control cells were treated with vehicles. Data is an average of three experimental repeats.

Figure 4.

(A) Hispolon (HISP) at 250 μM IC50 concentration induced a significant reduction of cell viability in human androgen resistance PC-3 prostate cancer cells and augmented docetaxel (DTX) anti-prostatic activity when compared with untreated control cells and to PC-3 cells treated with DTX alone (112 nM). (B) No significant reduction in cell viability of RWPE-1 immortalized normal human prostate cells. Control cells were treated with vehicles. Data is an average of three experimental repeats.

Figure 5.

Percent caspase-3 activity in androgen-resistant PC-3 human prostate cancer cells treated singly with IC50 concentrations for docetaxel (DTX), calebin A (CBA), 3^′-hydroxypterostilbene (HPSB), tetrahydrocurcumin (THC) and Hispolon (HISP) or in combination with DTX for 24 hours. Staurosporine was used as a positive control set at 100%. Data is an average of three experimental repeats. All treatment induced significant caspase activity when compared with vehicle treatment (untreated −ve control). DTX combination with test compounds caused significant increase of caspase percent activity when compared with DTX alone. Vehicle treated PC-3 cells showed no significant caspase-3 activity.

Figure 6.

Representatives bright filed microscopic images showing apoptosis in PC-3 prostate cancer cells. Micrographs of PC-3 cells treated with: (A) media, (B) staurosporine (positive control), (C) docetaxel (DTX), (D) tetrahydrocurcumin (THC), (E) hispolon (HISP), (F) 3^′-hydroxypterostilbene (HPSB), and (G) calebin A (CBA). Drugs were used at IC50 concentrations. Scale bars = 200 μm.

Figure 7.

Docetaxel (DTX) and four natural compounds caused significant inhibition of TNFα–NF-kB activation (colorimetric assay) when used singly (A) or when used in combination (B) each at IC₅₀ concentrations for 24 hours. Calebin A (CBA), 3^′-hydroxypterostilbene (HPSB), tetrahydrocurcumin (THC) a hispolon (HISP), and DTX significantly inhibited TNFα-induced NF-kB production in PC-3 human prostate cancer cells when compared with positive controls (P < 0.0001). Cells treated with TNF-α alone were considered positive controls (first and second bars from the left). CBA, HPSB, THC, and HISP were superior to DTX in inhibiting NF-kB. Data are an average of three experimental repeats.