Anti-cancer effects of novel flavonoid vicenin-2 as a single agent and in synergistic combination with docetaxel in prostate cancer

Abstract

The present study was conducted to determine the efficacy of novel flavonoid vicenin-2 (VCN-2), an active constituent of the medicinal herb Ocimum Sanctum Linn or Tulsi, as a single agent and in combination with docetaxel (DTL) in carcinoma of prostate (CaP). VCN-2 effectively induced anti-proliferative, anti-angiogenic and pro-apoptotic effect in CaP cells (PC-3, DU-145 and LNCaP) irrespective of their androgen responsiveness or p53 status. VCN-2 inhibited EGFR/Akt/mTOR/p70S6K pathway along with decreasing c-Myc, cyclin D1, cyclin B1, CDK4, PCNA and hTERT in vitro. VCN-2 reached a level of 2.6±0.3μmol/l in serum after oral administration in mice which reflected that VCN-2 is orally absorbed. The i.v. administration of docetaxel (DTL), current drug of choice in androgen-independent CaP, is associated with dose-limiting toxicities like febrile neutropenia which has lead to characterization of alternate routes of administration and potential combinatorial regimens. In this regard, VCN-2 in combination with DTL synergistically inhibited the growth of prostate tumors in vivo with a greater decrease in the levels of AR, pIGF1R, pAkt, PCNA, cyclin D1, Ki67, CD31, and increase in E-cadherin. VCN-2 has been investigated for radioprotection and anti-inflammatory properties. This is the first study on the anti-cancer effects of VCN-2. In conclusion, our investigations collectively provide strong evidence that VCN-2 is effective against CaP progression along with indicating that VCN-2 and DTL co-administration is more effective than either of the single agents in androgen-independent prostate cancer.

Figure 1. Anti-proliferative and pro-apoptotic effect of VCN-2 in CaP

Colony-forming assay was performed and the colonies were counted using Innotech Alpha Imager HP [23]. * p < 0.001 compared with control (panel A). For in-situ caspase-cleavage assay, the cells were grown on cover-slips and treated with 50 μM VCN-2 for 24 h and caspase-cleavage was measured using APO LOGIX™ carboxyfluorescein (FAM) caspase detection kit. Activated Caspase-positive cells appeared fluorescing green and pink when co-stained with DAPI (panel B). For detection of early and late events of apoptosis, Annexin V staining, TUNEL apoptotic assay and Hoechst staining were performed as described in Methods. Fluorescence was examined using Zeiss LSM 510 META laser-scanning fluorescence microscope. Apoptotic cells are green in Annexin V staining (membrane flipping), in TUNEL assay (DNA breaks revealed by end labeling), green fluorescence represents apoptotic cells, whereas blue in Hoechst staining (chromatin clumping) (panel C).

Figure 2. Effect of VCN-2 on angiogenesis, migration and cell cycle progression

The impact of VCN-2 on angiogenic sprout formation (arrows indicate borders new angiogenic vascular sprouts) in mice aortic rings cultured in Matrigels (panel A). VEGF-expression in control and 50 μM VCN-2 treated cells was performed by ELISA kit according to the manufacturer instructions (R&D Systems Inc., Minneapolis, MN) (panel B). Effect of VCN-2 on migration of LNCaP and PC3 cells in wound healing assay (panel C). Inhibitory effect of VCN-2 on cell cycle distribution was determined by FACS analysis. The experiment was repeated three times and similar results were obtained (panel D).

Figure 3. Effect of VCN-2 on apoptotic, proliferative and tumor suppressor proteins in CaP

Androgen-dependent (LNCaP) and androgen-independent (PC3) control and 50 μM VCN-2 treated cells were processed for Western-blot for PARP-cleavage and apoptosis regulatory proteins Bcl2 and Bax along with fibronectin and E-cadherin using specific antibodies (panel A). Western-blot analysis of the activation of pEGFR (Y¹⁰⁶⁸), pAkt (S⁴⁷³) and pP70S6K (S^240-244) in LNCaP and PC3 cells following VCN-2 treatment (panel B). Western-blot for the levels of cell-cycle regulatory proteins cyclin B1, cyclin D1, CDK4, PCNA and hTERT (panel C). Western-blot for oncoprotein C-Myc, and tumor suppressors Rb, pRb (S⁷⁸⁰) p53 and p21 (panel D). Membranes were stripped and reprobed for GAPDH as a loading control.

Figure 4. Effect of VCN-2 and DTL co-treatment on the survival, clonogenic potential, AR, IGF-1R and Akt activation in vitro cell cultures

The impact of VCN-2 (5 μM and 10 μM) and DTL (5 nM and 10 nM), alone and in combination, on the cell death as determined by MTT assay [26] (*CI<1, Chou-Talalay test, p<0.001, panel A) and on clonogenic potential as determined by colony-formation assay (*CI<1, Chou-Talalay test, p<0.004, panel B). Effect of VCN-2 and DTL on the regulation of AR in LNCaP cells by Western-blot against anti-AR IgG. Bars represent densitometry analysis (panel C). Effect of VCN-2 on IGF-1R signaling: Effect of VCN-2 on the regulation of constitutive and IGF-activated levels of pIGF1R (Y¹¹³¹) and down-stream pAkt (S⁴⁷³) in AR negative PC3 cells. The cells were incubated with indicated doses of VCN-2 for 24 h followed by incubation with 25 ng/ml IGF-1 for 30 min and subjected to immunobloting. (panel D).

Figure 5. Effect of oral administration of VCN-2 alone and in combination with DTL on tumor regression of PC3 xenografts

The nu/nu nude mice were injected with 2 × 10⁶ PC3 cells suspensions in 100 μl of PBS, subcutaneously into one flank and divided in to four groups. Treatment consisted of corn oil (control), VCN-2 (1 mg/kg b.w.), DTL (0.01 mg/kg b.w.), and both, VCN-2 and DTL, in 100 μl by oral gavage alternate day. Experimental details are given in the Methods section. Animals were examined daily for signs of tumor growth and body weights were recorded (panel A). Photographs of animals were taken at day 1, day 10, day 20, day 40, and day 60 after subcutaneous injection, are shown for all groups (supplementary data). Weights and photographs of tumors were also taken at day 60. * P < 0.001 for VCN-2 and DTL co-administration when compared to control as well as either of the single drug treatment (panel B). Tumors were measured in two dimensions using calipers and time-course analysis of tumor regression was performed during the study (panel C).

Figure 6. Histopathologic analyses of the markers of proliferation, angiogenesis and differentiation in tumor sections along with analyses of signaling proteins in tumor tissue lysates after VCN-2 treatment

Control, VCN-2 alone, DTL alone and VCN-2+DTL treated PC3 CaP bearing nude mice tumor sections were used for histopathologic analyses. Presented are H & E stained sections, IHC analyses for Ki67 expression (marker of cellular proliferation), CD31 (angiogenesis marker) and E-cadherin (tumor suppressor) from tumors in mice of control and experimental groups. Immuno-reactivity is evident as a dark brown stain. Sections were counterstained with Hematoxylin (blue). Photomicrographs at 40x magnification were acquired using Olympus Provis AX70 microscope. Percent staining was determined by measuring positive immuno-reactivity per unit area. Arrows represent the area for positive staining for an antigen. The intensity of antigen staining was quantified by digital image analysis. Statistical significance of difference was determined by two-tailed Student's t test. Bars represent mean ± S.E. (n = 5); * p<0.001 when compared to control (panel A). Western-blot Analysis of signaling proteins in tumor tissue lysates in control, VCN-2 alone, DTL alone as well as VCN-2 and DTL treated groups (panel B).