Hina Kausar

Withaferin A induces p53-dependent apoptosis by repression of HPV oncogenes and upregulation of tumor suppressor proteins in human cervical cancer cells

Cervical cancer is caused by human papilloma virus (HPV) expressing E6 and E7 oncoproteins, which are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Repression of HPV oncoproteins would therefore result in reactivation of tumor suppressor pathways and cause apoptosis in cancer cells. Withaferin A (WA), the active component of the medicinal plant Withania Somnifera, has exhibited inhibitory effects against several different cancers. We examined the activity of WA on human cervical cancer cells in vitro and in vivo. WA potently inhibited proliferation of the cervical cancer cells, CaSki (IC(50) 0.45 ± 0.05 μM). Mechanistically, WA was found to (i) downregulate expression of HPV E6 and E7 oncoproteins, (ii) induce accumulation of p53, (iii) increase levels of p21(cip1/waf1) and its interaction with proliferating cell nuclear antigen (PCNA), (iv) cause G(2)/M cell cycle arrest, associated with modulation of cyclin B1, p34(cdc2) and PCNA levels, (v) decrease the levels of STAT3 and its phosphorylation at Tyr(705) and Ser(727) and (vi) alter expression levels of p53-mediated apoptotic markers-Bcl2, Bax, caspase-3 and cleaved PARP. In vivo, WA resulted in reduction of nearly 70% of the tumor volume in athymic nude mice with essentially similar trend in the modulation of molecular markers as in vitro. This is the first demonstration indicating that WA significantly downregulates expression of HPV E6/E7 oncogenes and restores the p53 pathway, resulting in apoptosis of cervical cancer cells. Together, our data suggest that WA can be exploited as a potent therapeutic agent for the treatment and prevention of cervical cancer without deleterious effects.

Hsp27 Regulates Akt Activation and Polymorphonuclear Leukocyte Apoptosis by Scaffolding MK2 to Akt Signal Complex

We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117–128) on Akt as an Hsp27 binding region. Deletion of amino acids 117–128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate AktΔ117–128 mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

Antioxidant and Antiproliferative Activities of Anthocyanin/Ellagitannin-Enriched Extracts From Syzygium cumini L. (Jamun, the Indian Blackberry)

Colored fruits, particularly berries, are highly chemoprotective because of their antioxidant, antiproliferative, and antiinflammatory activities. We report the cancer chemoprotective potential of Syzygium cumini L., commonly known as jamun or Indian blackberry. Anthocyanins and other polyphenolics were extracted with acidic ethanol and enriched by amberlite XAD7/HP20 (1:1). The pulp powder was found to contain 0.54% anthocyanins, 0.17% ellagic acid/ellagitannins, and 1.15% total polyphenolics. Jamun seed contained no detectable anthocyanins but had higher amounts of ellagic acid/ellagitannins (0.5%) and total polyphenolics (2.7%) than the pulp powder. Upon acid hydrolysis, the pulp extract yielded 5 anthocyanidins by HPLC: malvidin (44.4%), petunidin (24.2%), delphinidin (20.3%), cyanidin (6.6%), and peonidin (2.2%). Extracts of both jamun pulp (1,445 ± 64 μmol of trolox equivalent (TE)/g) and seeds (3,379 ± 151 μM of TE/g) showed high oxygen radical absorbance capacity. Their high antioxidant potential was also reflected by 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)- and 2,2-diphenyl-1-picrylhydrazyl-scavenging and ferrous ion-chelating activities. We also analyzed antiproliferative activity of jamun extracts against human lung cancer A549 cells. The hydrolyzed pulp and seed extracts showed significant antiproliferative activity. However, unhydrolyzed extracts showed much less activity. These data showed that in addition to 5 anthocyanidins, jamun contains appreciable amounts of ellagic acid/ellagitannins, with high antioxidant and antiproliferative activities.

Interplay between Akt and p38 MAPK pathways in the regulation of renal tubular cell apoptosis associated with diabetic nephropathy

Hyperglycemia induces p38 MAPK-mediated renal proximal tubular cell (RPTC) apoptosis. The current study hypothesized that alteration of the Akt signaling pathway by hyperglycemia may contribute to p38 MAPK activation and development of diabetic nephropathy. Immunoblot analysis demonstrated a hyperglycemia-induced increase in Akt phosphorylation in diabetic kidneys at 1 mo, peaking at 3 mo, and dropping back to baseline by 6 mo. Immunohistochemical staining with anti-pAkt antisera localized Akt phosphorylation to renal tubules. Maximal p38 MAPK phosphorylation was detected concomitant with increase in terminal uridine deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity in 6-mo diabetic kidneys. Exposure of cultured RPTCs to high glucose (HG; 22.5 mM) significantly increased Akt phosphorylation at 3, 6, and 9 h, and decreased thereafter. In contrast, p38 MAPK phosphorylation was detected between 9 and 48 h of HG treatment. Increased p38 MAPK activation at 24 and 48 h coincided with increased apoptosis, demonstrated by increased caspase-3 activity at 24 h and increased TUNEL-positive cells at 48 h of HG exposure. Blockade of p38 cascade with SB203850 inhibited HG-induced caspase-3 activation and TUNEL-positive cells. Overexpression of constitutively active Akt abrogated HG-induced p38 MAPK phosphorylation and RPTC apoptosis. In addition, blockade of the phosphatidylinositol-3 kinase/Akt pathway with LY294002 and silencing of Akt expression with Akt small interfering RNA induced p38 MAPK phosphorylation in the absence of HG. These results collectively suggest that downregulation of Akt activation during long-term hyperglycemia contributes to enhanced p38 MAPK activation and RPTC apoptosis. Mechanism of downregulation of Akt activation in 6-mo streptozotocin diabetic kidneys was attributed to decreased Akt-heat shock protein (Hsp) 25, Akt-p38 interaction, and decreased PTEN activity. Thus PTEN or Hsp25 could serve as potential therapeutic targets to modulate Akt activation and control p38 MAPK-mediated diabetic complications.

Cucurbitacin B potently suppresses non-small-cell lung cancer growth: Identification of intracellular thiols as critical targets

Cucurbitacin B (CuB), has recently emerged as a potent anticancer agent; however, its efficacy in non-small-cell lung cancer (NSCLC) and the mechanism(s) initiating its biological effects remain largely unclear. In this study, CuB potently suppressed the growth of four NSCLC cells (H1299, A549, HCC-827 and H661) in vitro and the highly aggressive H1299 xenograft in vivo. CuB significantly altered the actin cytoskeletal assembly, induced G2/M cell-cycle arrest and mitochondrial apoptosis through the modulation of several key molecular targets mediating the aforementioned processes. Interestingly, all cellular effects of CuB were completely attenuated only by the thiol antioxidant N-acetylcysteine (NAC). Furthermore, pretreatment with glutathione synthesis inhibitor butithione-sulfoxime (BSO), significantly exacerbated CuBs cytotoxic effects. To this end, cells treated with CuB revealed a rapid and significant decrease in the levels of protein thiols and GSH/GSSG ratio, suggesting disruption of cellular redox balance as the primary event in CuBs cytotoxic arsenal. Using UV and FTICR mass spectrometry we also demonstrate for the first time a physical interaction of CuB with NAC and GSH in a cell-free system suggesting that CuB interacts with and modulates cellular thiols to mediate its anti-cancer effects. Collectively, our data sheds new light on the working mechanisms of CuB and demonstrate its therapeutic potential against NSCLC.

Exosomal formulation enhances therapeutic response of celastrol against lung cancer

Celastrol (CEL), a plant-derived triterpenoid, is a known inhibitor of Hsp90 and NF-κB activation pathways and has recently been suggested to be of therapeutic importance in various cancers. However, the molecular mechanisms of celastrol-mediated effects in lung cancer are not systematically studied. Moreover, it suffers from poor bioavailability and off-site toxicity issues. This study aims to study the effect of celastrol loaded into exosomes against two non-small cell-lung carcinoma (NSCLC) cell lines and explore the molecular mechanisms to determine the proteins governing the cellular responses. We observed that celastrol inhibited the proliferation of A549 and H1299 NSCLC cells in a time- and concentration-dependent manner as indexed by MTT assay. Mechanistically, CEL pre-treatment of H1299 cells completely abrogated TNFα-induced NF-κB activation and upregulated the expression of ER-stress chaperones Grp 94, Grp78, and pPERK. These changes in ER-stress mediators were paralleled by an increase in apoptotic response as evidenced by higher annexin-V/PI positive cells evaluated by FACS and immunoblotting which showed upregulation of the ER stress specific pro-apoptotic transcription factor, GADD153/CHOP and alteration of Bax/Bcl2 levels. Exosomes loaded with CEL exhibited enhanced anti-tumor efficacy as compared to free CEL against lung cancer cell xenograft. CEL did not exhibit any gross or systemic toxicity in wild-type C57BL6 mice as determined by hematological and liver and kidney function test. Together, our data demonstrate the chemotherapeutic potential of CEL in lung cancer and that exosomal formulation enhances its efficacy and reduces dose related toxicity.

Controlled-release systemic delivery - a new concept in cancer chemoprevention.

Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.

Controlled systemic delivery by polymeric implants enhances tissue and plasma curcumin levels compared with oral administration.

Curcumin possesses potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ∼2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ∼3.3 ng/ml on day 1, which dropped to ∼0.2 ng/ml after 3 months, whereas only 0.2-0.3 ng/ml concentration was observed from 4-12 days with diet and was undetected subsequently. Almost 10-fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1 ± 7.3 vs 2.7 ± 0.8 ng/g) and were still significant even after 90 days (7.7 ± 3.8 vs 2.2 ± 0.8 ng/g). Although curcumin levels were similar in liver from both the routes (∼25-30 ng/g from day 1-4 and ∼10-15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ∼28-fold lower doses at 90 days. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.

Lung cancer inhibitory activity of dietary berries and berry polyphenolics

BACKGROUND: Blueberry (BB) and black raspberry (BRB) have been shown to be chemopreventive against estrogenmediated breast cancer in pre-clinical studies. However, therapeutic efficacy of these berries against lung cancer is not known. METHODS: In this study we investigated i) relative efficacy of individual anthocyanidins vs. respective anthocyanins, ii) relative antiproliferative activity of mixture of anthocyanidins compared to individual anthocyanidins, iii) antitumor activity of dietary BB, iv) Tumor inhibitory activity of diet supplemented with BB, alone and in combination with BRB, against lung tumor xenograft using nude mice, and finally, v) the efficacy of select polyphenolics present in BB and BRB against lung tumor xenograft. RESULTS: Our findings indicated that individual anthocyanidins (aglycones) were significantly more potent (2-3 fold lower IC50) in inhibiting the non-small-cell lung cancer (NSCLC) cell growth vs. respective anthocyanins (glycones). Further, anthocyanidins mixture at equimolar concentrations exhibited synergistic antiproliferative activity vs. individual anthocyanidins. When tested against NSCLC (A549 and H1299) cells in nude mice, dietary BB (7.5%, w/w) showed >40% reduction in tumor volume against H1299 xenografts. The maximal growth inhibition occurred with 5% BB dose, with no additional protection occurring at a higher dose (7.5%). However, somewhat lower protection was found when the BB diet initiated prior to tumor cell inoculation. The mixture of BB (5%, w/w) and BRB (2.5%) resulted in higher inhibition of tumor growth vs. BB alone (71% vs 42%). Likewise, a combination of delphinidin (bioactive of BB) and punicalagins (a bioactive of BRB, which gets converted to ellagic acid in vivo) showed higher tumor growth inhibition compared to delphinidin. CONCLUSIONS: The therapeutic effects of the berries and berry polyphenolics observed against lung cancer in this study are highly encouraging. Further investigation into the mechanism of action of the combinations of the berry bioactives will be valuable for clinical use of this potent natural product against lung cancer.

Curcumin Implants, Not Curcumin Diet, Inhibit Estrogen-Induced Mammary Carcinogenesis in ACI Rats

Curcumin is widely known for its antioxidant, anti-inflammatory, and antiproliferative activities in cell-culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvent oral bioavailability issues, and tested their potential against 17β-estradiol (E2)–mediated mammary tumorigenesis. Female Augustus Copenhagen Irish (ACI) rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2 cm; 200 mg each; 20% drug load) 4 days before grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Curcumin implants were changed after 4.5 months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months, and after the tumor incidence reached >80% (∼6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2 ± 1 vs. 5 ± 3; P = 0.001) and tumor volume (184 ± 198 mm3 vs. 280 ± 141 mm3; P = 0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity, whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in the presence of E2. Because CYP1A and CYP3A4 metabolize most of the E2 to its noncarcinogenic 2-OH metabolite, and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by high-performance liquid chromatography showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered. Cancer Prev Res; 7(4); 456–65. ©2014 AACR.