Contributed equally
In the present study, we aimed to investigate the anticancer properties of Theracurmin®, a novel form of the yellow curry pigment curcumin, as well as explore the molecular mechanisms of the potential anticancer effects of Theracurmin® on human prostate cancer and bladder cancer cells
Recently, complementary and alternative medicine (CAM) has received great interest among cancer patients as an alternative therapeutic method (
However, the clinical efficacy of curcumin is still limited, which is most likely due to its low bioavailability (
Human prostate cancer cell lines (LNCaP, PC3, and DU145) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). We used RPMI-1640 medium (Welgene, Daegu, Korea) for LNCaP and PC3, and Dulbecco's modified Eagle's medium (DMEM; Welgene) for DU145 cells as the basal culture medium. Human bladder cancer cell lines (T24, 253J, and HTB9) were also purchased from ATCC. To establish a cisplatin-resistant cell line (T24R2), we performed serial desensitization of T24 cells as previously described (
To examine the cell proliferation, we used the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA). Cells (2×103 cells/well) were seeded onto 96-well plates and incubated for 24 h. Cells were treated with either curcumin or Theracurmin® for 24, 48, and 72 h, and then 10
Cells (1×103 cells/well) were seeded onto 35-mm2 dishes and treated with either curcumin or Theracurmin® for 48 h. To form visible colonies, the cells were cultured for an additional 14 day in either curcumin- or Theracurmin®-free culture condition. After fixation with 10% neutral-buffered formalin solution, the samples were stained with 0.1% crystal violet solution (both from Sigma-Aldrich). Finally, the samples were photographed, and the number of visible colonies comprising more than 50 individual cells was determined by using a SZX7 stereomicroscope (Olympus, Tokyo, Japan).
Cells (3×105 cells/well) were plated on 60-mm2 dishes and incubated with either curcumin or Theracurmin® for 48 h. The cells were fixed in 70% ethanol and reacted with RNase A (10
To extract total proteins, the cells were lysed with radio-immunoprecipitation assay buffer [50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and 1 mM phenylmethylsulfonyl fluoride]. After measuring protein concentrations of each sample using the BCA protein assay kit (Pierce, Rockford, IL, USA), samples were prepared with equal amounts of protein in 1X SDS buffer. Protein samples were electrophoresed on SDS-PAGE gels and transferred onto poly-vinylidene difluoride membranes (Millipore, Billerica, MA, USA). After the samples were blocked with 5% (w/v) non-fat dry milk at room temperature for 1 h, the blots were incubated with primary antibodies overnight at 4°C. Primary antibodies used in the present study were anti-cleaved caspase-3, -8, -9, poly(adenosine diphosphate-ribose) polymerase (PARP), cytochrome
Three independent experiments were performed in triplicate, and the data are presented as mean ± standard error of the mean (SEM). The statistical analysis was conducted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). We considered data with p-values <0.05 as significant, as determined by Turkey's multiple range tests.
To examine the cytotoxic effects of Theracurmin® on human prostate cancer cells, we treated cells with this agent for 24, 48 and 72 h and examined the cell viability using the CCK-8 assay. Cell viability of the PC3, DU145 and LNCaP cells was significantly decreased in a dose- and time-dependent manner after Theracurmin® treatment (
These results indicate that Theracurmin® and curcumin have similar anticancer effects on human prostate cancer cells in both androgen-dependent and -independent cells in a dose- and time-dependent manner.
To further examine the molecular mechanisms of the anticancer activities of Theracurmin® in human prostate cancer cells, we investigated the expression patterns of molecules associated with apoptosis, as well as the cell cycle distribution patterns after Theracurmin® treatment. Western blot analysis showed that pro-apoptotic proteins (cleaved PARP, caspase-3, -8, and -9, cytochrome
These findings suggest that Theracurmin® and curcumin treatment led to the reduction in cell viability and clonogenic potential via the induction of apoptotic cell death in human prostate cancer cells. Partly, cell cycle disturbance, such as G2/M arrest, played an important role in the cytotoxicity of Theracurmin® treatment in both androgen-dependent and -independent cells.
We next examined the cytotoxic effects of Theracurmin® on human bladder cancer cell lines (T24, 293J, and HTB9) as well as on the cisplatin-resistant cell line, T24R2. Similar to the results using prostate cancer cells, Theracurmin® treatment markedly reduced the viability of the bladder cancer cell lines in a dose- and time-dependent manner (
We revealed that Theracurmin® treatment also significantly diminished the clonogenic proliferation of human bladder cancer cells in a dose-dependent manner (
To explore the molecular aspects of the anticancer effects of Theracurmin® in human bladder cancer cells, we examined the expression patterns of apoptosis- and cell cycle-regulating molecules after adding Theracurmin®. Similar to the findings in prostate cancer cells, the pro-apoptotic proteins (cleaved PARP, caspase-3, -8, and -9, cytochrome
Prostate and bladder cancer are usually diagnosed in older men, who may be vulnerable to the side effects from first-line chemotherapeutic agents, such as docetaxel and cisplatin. In this context, natural compounds can be beneficial as an alternative therapeutic approach for treating these urological cancers (
Although many preclinical studies have shown curcumin to be a promising anticancer agent in urological cancer, its poor bioavailability is a major drawback to its clinical application for cancer patients (
Despite these promising results showing Theracurmin® to be a safe and effective anticancer agent, no proof-of-concept studies have been carried out in urological cancer. In this study, we first revealed that Theracurmin® significantly reduced the cell viability in human prostate cancer and bladder cancer cells, particularly showing comparable anticancer efficacy with curcumin. Notably, the anticancer effects of Theracurmin® were exerted by inducing apoptotic cell death and cell cycle disturbance in these urological cancer cells. Similar to our findings, Kamat
Apoptosis and cell cycle arrest, such as in the S or G2/M phases, are regarded as the cellular response pathways following diverse stimuli, including DNA damage (
Mechanistically, curcumin has been shown to modulate many molecular targets, including transcription factors, oncogenic growth factors, and inflammatory cytokines with relevant multiple signaling pathways, resulting in inhibition of cell proliferation, invasion and metastasis (
However, our study did not provide a more comprehensive data for the molecular mechanisms of the anticancer activities of Theracurmin® in human prostate and bladder cancer cells. Our data only demonstrated the phenotypic changes of human prostate cancer and bladder cancer cells after Theracurmin® and curcumin treatment, which is the major limitation of our study. In order to provide more concrete evidence, we should explore the relevant signaling pathways. More importantly, this study only provided
In summary, we provides
The present study was supported by a grant from Handok Pharmaceuticals in Korea. The funders had no role in study design, experiments, data collection and analysis, preparation of the manuscript, or the decision to publish.
Anticancer effects of Theracurmin® and curcumin treatment in human prostate cancer cells. Cell viability assessments for determining cytotoxic effects of (A) Theracurmin® or (B) curcumin treatment in human prostate cancer cell lines (PC3, DU145, and LNCaP). The cells were treated with different concentrations of each agent, ranging from 1 to 100
Theracurmin® and curcumin treatment induce apoptotic cell death and cell cycle dysregulation in human prostate cancer cells. (A) Western blot analysis for the expression of molecules associated with apoptosis (total PARP, cleaved PARP, caspase-3, caspase-8, caspase-9, Bcl-2, Bad, and cytochrome
Anticancer effects of Theracurmin® and curcumin treatment in human bladder cancer cells. Cell viability assessments for determining the cytotoxic effects of (A) Theracurmin® or (B) curcumin treatment in human prostate cancer cell lines (T24, 253J, HTB9, and cisplatin-resistant T24R2). The cells were treated with different concentrations of each agent, ranging from 1 to 100
Theracurmin® and curcumin treatments induce apoptotic cell death and cell cycle dysregulation in human bladder cancer cells. (A) Western blot analysis for the expression of molecules associated with apoptosis (total PARP, cleaved PARP, caspase-3, caspase-8, caspase-9, Bcl-2, Bad, and cytochrome