Concomitant Decrease In Bcl-2 Research Articles

Abstract 3725: Pre-clinical evaluation of prostate specific membrane antigen (PSMA) nanobioconjugate encapsulating green tea polyphenol EGCG for prostate cancer

Abstract For prostate cancer green tea constituent (-)-epigallocatechin-3-gallate (EGCG) has shown promise in preclinical, geographical and epidemiological studies and more importantly in many clinical trials. However, concerns related to stability, poor intestinal absorption and extensive systemic and enteric metabolism have hampered its use in the clinic. We introduced the idea of nanotechnology to enhance the outcome of chemoprevention with EGCG and coined the term ‘Nanochemoprevention' and later reported the efficacy of chitosan nanoparticles encapsulating EGCG for PCa prevention and therapy. Here, we evaluated an aptamer conjugated prostate targeting nanobioconjugate encapsulating EGCG in two relevant pre-clinical mouse models of human PCa. We developed chitosan nanobioconjugates encapsulating EGCG, surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the prostate specific membrane antigen (PSMA), henceforth called chit-EGCG-Apt. Mice implanted with androgen sensitive CWR22Rν1 cells and treated with Chit-EGCG-Apt showed significant inhibition in tumor growth that was also associated with decreased serum PSA levels as compared to native EGCG. Tumor tissues from mice treated with Chit-EGCG-Apt exhibited significant (i) induction of PARP cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and PCNA. We further observed inhibition of markers of angiogenesis, invasion and metastasis in tumor tissues in these mice. We next evaluated the efficacy of Chit-EGCG-Apt in the prostate specific Pten knockout mouse model that recapitulates disease progression as seen in humans. Longitudinal MRI analysis suggested significant inhibition of PCa development and tumor growth in these mice. We observed a significant inhibition in tumor growth as evaluated by genitourinary and prostate tissue weight. We further observed a significant decrease in serum IGF-I levels with concomitant increase in serum IGFBP-3 levels in these mice. We analyzed tumor tissues isolated from the mice and observed significant (i) induction of apoptosis, (ii) modulation of Pten/PI3K/Akt, MAPK pathway, IGF-1 signaling and iii) inhibition of markers associated with angiogenesis, invasion and metastasis. These data support our hypothesis that target-specific nanobioconjugates enhance efficacy through boosting bioavailability, thus leading to a significant potential for possible clinical outcome. Targeted nano-encapsulation approach could be viewed as “precision chemoprevention” for PCa patients that are considered low-risk and put on active surveillance. Citation Format: Imtiaz A. Siddiqui, Vaqar M. Adhami, Islam Rady, Hasan Mukhtar. Pre-clinical evaluation of prostate specific membrane antigen (PSMA) nanobioconjugate encapsulating green tea polyphenol EGCG for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3725.

Abstract 5263: Prostate-specific membrane antigen (PSMA)-targeting nanobioconjugate-encapsulated green tea polyphenol EGCG for prostate cancer prevention and therapy

Abstract Initially we introduced the idea of employing nanotechnology to improve the outcome of natural agents in chemoprevention and coined the term ‘Nanochemoprevention’. Recently we reported the efficacy of chitosan nanoparticles encapsulating (-)-epigallocatechin-3-gallate (EGCG) for prostate cancer (PCa) prevention and therapy. Here we employed the concept of targeted uptake of therapeutic nanoparticles, a potentially powerful technology to enhance the efficacy of EGCG for PCa management. We developed chitosan nanobioconjugates encapsulating EGCG and surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the PSMA, henceforth called chit-EGCG-Apt. Cellular binding and uptake of the fluorescent nanosystem was detected suggesting PSMA specific internalization and accumulation. Further, we observed that chit-EGCG-Apt lead to a PSMA specific increase of anti-proliferative activity in PCa cells, with a better efficacy in PSMA positive cells (LNCaP and C4-2) than in PSMA negative (PC3 and DU145) cells and a decrease in IC50 by at least half the dose of the native agent. Further, we also observed a significant inhibition in the colony formation potency of PCa cells when treated with chit-EGCG-Apt as compared to the native EGCG with the effects more distinct in PSMA positive cells. Next, we established that EGCG in chit-EGCG-Apt retains its mechanistic identity and observed that the treatment resulted in significant (i) shift in Bax/Bcl-2 ratio in favor of apoptosis, (ii) increase in PARP cleavage, (iii) inhibition in caspase 3, 7, 8 and 9, and other key regulators of apoptosis, (iv) modulation of death receptor pathway proteins, and (v) cyclin dependent kinase inhibitors in addition to arrest of the cells in S phase of the cell cycle. Importantly, these effects were only visible at higher doses of the native EGCG. The nanobioconjugate also demonstrated a significant inhibition of invasive potential of several PCa cells and 3-dimensional spheroid BME cell invasion assay. Further, chit-EGCG-Apt treated mice implanted with androgen sensitive CWR22R(nu)1 cells showed significant inhibition in tumor growth and decreased serum PSA levels reciprocating our in vitro findings. In tumor tissues of mice treated with Chit-EGCG-Apt there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases, and (iv) reduction in Ki-67 and proliferating cell nuclear antigen, as compared to native EGCG. We further observed inhibition of angiogenesis, invasion and metastasis markers in these tissues. Translation of these data to appropriate animal model systems could pave way for prostate cancer therapy in humans. Citation Format: Imtiaz A. Siddiqui, Dhruba J. Bharali, Rahime Jashari, Vaqar M. Adhami, Shaker A. Mousa, Hasan Mukhtar. Prostate-specific membrane antigen (PSMA)-targeting nanobioconjugate-encapsulated green tea polyphenol EGCG for prostate cancer prevention and therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5263.

Abstract 236: Antiproliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate encapsulated in chitosan nanoparticles on human prostate carcinoma cells

Abstract In preclinical animal models (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has shown excellent effects in preventing and/or treating many cancers including prostate cancer (PCa). Chemoprevention is a practical approach for cancer management however its applicability to humans has met with limited success due to several issues including its limited bioavailability. We recently employed the use of nanotechnology to improve the outcome of natural agents for cancer chemoprevention and termed the concept as ‘nanochemoprevention’. To demonstrate the proof-of-principle we encapsulated EGCG in PLA-PEG nanoparticles and showed that this formulation demonstrates greater than ten-fold dose advantage over non-encapsulated EGCG in human PCa PC3 cells both in vitro and in vivo. However, this nanoformulation is not suitable for oral delivery. We recently reported the synthesis, characterization and efficacy assessment of a nanotechnology-based formulation of chitosan encapsulating EGCG (chit-nanoEGCG) for treatment of PCa in a xenograft model. We now show the efficacy of chit-nanoEGCG in LNCaP (androgen responsive) and PC3 (androgen unresponsive) human PCa cells for induction of apoptosis and inhibition of cell growth. The effective dose of EGCG for inhibiting cell growth was reduced significantly when encapsulated in chitosan nanoparticles. The IC50 of chit-nanoEGCG was observed to be 4 μM as compared to 90 μM with native EGCG in PC3 cells. For LNCaP cells the IC50 of chit-nanoEGCG was observed to be 1 μM as compared to 45 μM of native agent. Further, we also observed a significant inhibition in the colony formation potential of these cells when treated with low doses of chit-nanoEGCG (2-4 μM) at which native EGCG had no effect. Our next goal was to determine if EGCG encapsulated in this nanoformulation retains its mechanistic identity. Treatment of LNCaP and PC3 with chit-nanoEGCG (1 and 2 μM) and (4 and 6 μM), respectively, for 48 hrs as compared with native EGCG (40 and 50 μM) and (80 and 100 μM) resulted in significant: i) induction of poly (ADP-ribose) polymerases cleavage (PARP), ii) increase of protein expression of Bax with concomitant decrease in Bcl2 and, iii) activation of cleaved caspases 3,7,8,9. To further confirm the induction of apoptosis, we performed annexin/PI staining and observed significant presence of annexin positive cells in chit-nanoEGCG treated PC3 and LNCaP cells. Further, we also observed that chit-nano EGCG treatment resulted in arrest of the cells in S phase of the cell cycle and cell cycle-regulatory molecules operative in the G1/S phase of the cell cycle were significantly modulated. All these effects were seen at much lower doses in chit-nanoEGCG treated cells as compared to the native agent. Collectively, chit-nanoEGCG could prove useful for preventing and/or treating human PCa. Citation Format: Sameh M. Shabana, Imtiaz A. Siddiqui, Naghma Khan, Vaqar M. Adhami, Qateeb A. Khan, Shaker A. Mousa, Ahmed E. Hagras, Hasan Mukhtar, Mohamed A. Elmissiry. Antiproliferative and proapoptotic effects of (-)-epigallocatechin-3-gallate encapsulated in chitosan nanoparticles on human prostate carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2014-236

Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model

In preclinical animal models, several phytochemicals have shown excellent potential to be used as effective agents in preventing and treating many cancers. However, the limited bioavailability of active agents could be one reason for their restricted usefulness for human consumption. To overcome this limitation, we recently introduced the concept of nanochemoprevention by encapsulating useful bioactive food components for their slow and sustained release. Here, we report the synthesis, characterization and efficacy assessment of a nanotechnology-based oral formulation of chitosan nanoparticles encapsulating epigallocatechin-3-gallate (Chit-nanoEGCG) for the treatment of prostate cancer (PCa) in a preclinical setting. Chit-nanoEGCG with a size of <200nm diameter and encapsulating EGCG as determined by dynamic light scattering and transmission electron microscope showed slow release of EGCG in simulated gastric juice acidic pH and faster release in simulated intestinal fluid. The antitumor efficacy of Chit-nanoEGCG was assessed in subcutaneously implanted 22Rν1 tumor xenografts in athymic nude mice. Treatment with Chit-nanoEGCG resulted in significant inhibition of tumor growth and secreted prostate-specific antigen levels compared with EGCG and control groups. In tumor tissues of mice treated with Chit-nanoEGCG, compared with groups treated with EGCG and controls, there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases and (iv) reduction in Ki-67 and proliferating cell nuclear antigen. Through this study, we propose a novel preventive and therapeutic modality for PCa using EGCG that addresses issues related to bioavailability.

Aptamer-conjugated and doxorubicin-loaded unimolecular micelles for targeted therapy of prostate cancer

In the absence of effective therapy for prostate cancer, there is an immense need for developing improved therapeutic options for the management of this disease. This study has demonstrated that aptamer-conjugated unimolecular micelles can improve the in vivo tumor biodistribution of systemically administered anti-cancer drugs in prostate cancer expressing prostate-specific membrane antigen (PSMA). The aptamer-conjugated unimolecular micelles were formed by individual hyperbranched polymer molecules consisting of a hyperbranched H40 polymer core and approximately 25 amphiphilic polylactide–poly(ethlyene glycol) (PLA–PEG) block copolymer arms (H40-PLA-PEG-Apt). The unimolecular micelles with an average hydrodynamic diameter of 69 nm exhibited a pH-sensitive and controlled drug release behavior. The targeted unimolecular micelles (i.e., DOX-loaded H40-PLA-PEG-Apt) exhibited a much higher cellular uptake in PSMA positive CWR22Rν1 prostate carcinoma cells than non-targeted unimolecular micelles (i.e., DOX-loaded H40-PLA-PEG), thereby leading to a significantly higher cytotoxicity. The DOX-loaded unimolecular micelles up-regulated the cleavage of PARP and Caspase 3 proteins and increased the protein expression of Bax along with a concomitant decrease in Bcl2. These micelles also increased the protein expression of cell cycle regulation marker P21 and P27. In CWR22Rν1 tumor-bearing mice, DOX-loaded H40-PLA-PEG-Apt micelles (i.e., targeted) also exhibited a much higher level of DOX accumulation in the tumor tissue than DOX-loaded H40-PLA-PEG micelles (i.e., non-targeted). These findings suggest that aptamer-conjugated unimolecular micelles may potentially be an effective drug nanocarrier to effectively treat prostate cancer.

ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation

IntroductionThe present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers.MethodsWe investigated the effects of ABL-N on the induction of apoptosis in human breast cancer cells and further examined the underlying mechanisms. Moreover, tumor growth inhibition of ABL-N was done in xenograft models.ResultsABL-N induced the activation of caspase-3 in estrogen receptor (ER)-negative cell lines MDA-MB-231 and MDA-MB-468, as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP). Pretreatment of cells with pan-caspase inhibitor z-VAD-fmk or caspase-3-specific inhibitor z-DEVD-fmk inhibited ABL-N-induced apoptosis. ABL-N treatment also resulted in an increase in the expression of pro-apoptotic members (Bax and Bad) with a concomitant decrease in Bcl-2. Furthermore, c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) were activated in the apoptosis induced by ABL-N and JNK-specific inhibitor SP600125 and JNK small interfering RNA (siRNA) antagonized ABL-N-mediated apoptosis. However, the p38-specific inhibitor SB203580 had no effect upon these processes. Moreover, neither of the caspase inhibitors prevented ABL-N-induced JNK activation, indicating that JNK is upstream of caspases in ABL-N-initiated apoptosis. Additionally, in a nude mice xenograft experiment, ABL-N significantly inhibited the tumor growth of MDA-MB-231 cells.ConclusionsABL-N induces apoptosis in breast cancer cells through the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment causes a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.

Radiosensitization of HT‐29 cells and xenografts by the nitric oxide donor DETANONOate

Mechanisms of radioresistance in rectal cancer remain unclear. To determine mechanisms of radioresistance in rectal cancer cells and to assess the role of the nitric oxide donor DETANONOate as a radiosensitizing agent. Survival was determined by clonogenic assays, apoptosis by PARP-1 cleavage, and phenotypic differences by Western blot analysis. SCID mice bearing HT-29 xenografts were treated with ionizing radiation (IR) [2.0 Gy x 5], DETANONOate [0.4 mg/kg i.p.], or combination treatment. Colorectal cancer HT-29-p53-null cells were resistant and HCT-116-p53 wild-type cells sensitive to IR, which correlated with cleaved PARP-1. Increased levels of p21 occurred in HCT-116 cells, while Bcl-2 and survivin were elevated in HT-29 cells. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in DETANONOate-HT-29-treated versus control cells, which was accompanied by elevation of p21, p27, and BAX, and a concomitant decrease in Bcl-2. SCID mice bearing HT-29 xenografts demonstrated a 37.6%, 51.1%, and 70.1% inhibition in tumor growth in mice receiving IR, DETANONOate, and combination treatment versus control, respectively. Radioresistant HT-29 cells are p53-null and have substantially decreased levels of p21. DETANONOate radiosensitized HT-29 cells in vitro and in vivo by an additive effect in apoptosis.

Bromelain inhibits COX-2 expression by blocking the activation of MAPK regulated NF-kappa B against skin tumor-initiation triggering mitochondrial death pathway

Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple ( Ananas cosmosus), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of ∼65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-κB) was recorded, as phosphorylation and consequent degradation of Iκ Bα was blocked by bromelain. Also, bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by bromelain is a coherent approach in achieving chemoprevention.

Therapeutic intervention of experimental breast cancer bone metastasis by indole-3-carbinol in SCID-human mouse model

Several lines of experimental evidence have suggested that chemokine receptor CXCR4, a metastasis-promoting molecule, may play important roles in breast cancer bone metastasis. There is emerging evidence linking CXCR4 to matrix metalloproteinases (MMP) as well as their regulator nuclear factor-kappaB (NF-kappaB), a key transcription factor, which is known to activate metastasis-promoting molecules for many types of malignancies, including breast cancer. A recent study also showed that promoter region of CXCR4 has several NF-kappaB-binding sites, suggesting that there may be a cross-talk between CXCR4 and NF-kappaB. We have shown previously that indole-3-carbinol (I3C), a natural compound present in vegetables of the genus Brassica, can inhibit NF-kappaB in breast cancer cells. However, there are no reports in the literature showing any effect of I3C on CXCR4 expression in vitro and in vivo. We therefore examined whether I3C could inhibit bone metastasis of breast cancer by inhibiting CXCR4 and MMP-9 expression mediated via the inhibition of the NF-kappaB signaling pathway. Here, we have modified the severe combined immunodeficient (SCID)-human mouse model of experimental bone metastasis for use with the MDA-MB-231 breast cancer cell line. In this animal model, we found that I3C significantly inhibited MDA-MB-231 bone tumor growth, and our results were correlated with the down-regulation of NF-kappaB. Moreover, we found that I3C significantly inhibited the expression of multiple genes involved in the control of metastasis and invasion in vitro and in vivo, especially the expression of CXCR4 and MMP-9 along with pro-MMP-9, with concomitant decrease in Bcl-2 and increase in the proapoptotic protein Bax. From these results, we conclude that the CXCR4/NF-kappaB pathway is critical during I3C-induced inhibition of experimental breast cancer bone metastasis. These results also suggest that I3C could be a promising agent for the prevention and/or treatment of breast cancer bone metastasis in the future.