Papers by Mian Nasir Shahzad
Neoplasia, 2011
RNA interference holds tremendous potential as a therapeutic approach, especially in the treatmen... more RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.
Neoplasia, 2013
Chronic sympathetic nervous system activation results in increased angiogenesis and tumor growth ... more Chronic sympathetic nervous system activation results in increased angiogenesis and tumor growth in orthotopic mouse models of ovarian carcinoma. However, the mechanistic effects of such activation on the tumor vasculature are not well understood. Dopamine (DA), an inhibitory catecholamine, regulates the functions of normal and abnormal blood vessels. Here, we examined whether DA, an inhibitory catecholamine, could block the effects of
Cancer Research, 2011
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chronic stress is known ... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Chronic stress is known to promote tumor growth by activating the sympathetic nervous system (SNS) with resultant increases in norepinephrine (NE) and epinephrine (E). In contrast to NE and E, dopamine (DA) levels are low under chronic stress conditions. We have recently demonstrated that dopamine replacement in mice exposed to daily restraint stress can reverse the stimulatory effects of NE and E on ovarian cancer growth. Ovarian tumor tissues from stressed mice treated with dopamine showed significant increases in pericyte coverage of tumor microvessels. The focus of the current study was to examine the mechanisms by which dopamine treatment affected pericyte coverage of tumor endothelial cells under stress conditions. Female nude mice were subjected to chronic stress using the restraint-stress procedure. Tumor formation was induced by injecting the SKOV3 ip1 or HeyA8 ovarian cancer cells into the peritoneal cavity. Stressed and non-stressed mice were divided into four treatment groups: Control (PBS), DA, DA plus butaclamol (DR1 antagonist) and DA plus eticlopride (DR2 antagonist). Following therapy, harvested tumors were examined for microvessel density (MVD), vascular maturation (pericyte coverage) and proliferating cell nuclear antigen (PCNA). Expression of DA receptors (DR1-DR5) was analyzed by RT-PCR and Western blotting. Mice exposed to daily restraint stress showed 2-fold increased SKOV3ip1-tumor growth compared to non-stressed mice; treatment with DA alone resulted in 78% (p<0.01) decrease in tumor growth and 68% (p<0.01) decrease in MVD compared to control stressed mice. Similarly, DA/butaclamol combined treatment led to 71% (p<0.01) reduction in tumor growth and a 65% (p<0.01) decrease in MVD. Eticlopride in combination with DA reversed the inhibitory effects of DA on tumor growth. Furthermore, in stressed mice, daily dopamine treatment resulted in significant increase in pericyte coverage (51.4%; p<0.001) compared to controls. This effect was abrogated by butaclamol (44%; p<0.01), but not by eticlopride treatment. Similar results were obtained in stressed mice bearing HeyA8-tumors. In T10.5-pericyte like cells, treatment with DA, DA plus NE, DA agonist: [SKF38393][1] or PDGFBB increased significantly cell migration. No significant changes were noted with NE alone or DA plus butaclamol treatments. In MOEC, no significant changes in cell migration were observed under these experimental conditions. Collectively, our data indicate that DA, acting through DR1, could stimulate recruitment of pericytes to tumor endothelial cells, and promote vascular maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2011-406 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=SKF38393&atom=%2Fcanres%2F71%2F8_Supplement%2F406.atom
Molecular Cancer Therapeutics, 2010
This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therap... more This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therapy regimen of metronomic topotecan and pazopanib, a multireceptor tyrosine kinase inhibitor. In vitro (Western blot) and in vivo dose-finding experiments were done following pazopanib therapy in ovarian cancer models. Pazopanib and metronomic (daily) oral topotecan therapy was examined in an orthotopic model of ovarian cancer. Tumor weights, survival, and markers of the tumor microenvironment [angiogenesis (CD31 and pericyte coverage), proliferation (Ki-67), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling)] were analyzed by immunostaining following therapy. Pazopanib therapy reduced vascular endothelial growth factor receptor 2 (VEGFR-2) activity in vitro and vivo in a dose-dependent manner. Compared with control mice, pazopanib reduced tumor weight by 28% to 82% (P < 0.01 in the SKOV3ip1 model) and metronomic topotecan reduced tumor weight by 40% t...
Molecular Cancer Therapeutics, 2010
EphB4 is a transmembrane receptor tyrosine kinase that plays an important role in neural plastici... more EphB4 is a transmembrane receptor tyrosine kinase that plays an important role in neural plasticity and angiogenesis. EphB4 is overexpressed in ovarian cancer and is predictive of poor clinical outcome. However, the biological significance of EphB4 in ovarian cancer is not known and is the focus of the current study. Here, we examined the biological effects of two different methods of EphB4 targeting (a novel monoclonal antibody, EphB4-131 or siRNA) using several ovarian cancer models. EphB4 gene silencing significantly increased tumor cell apoptosis and decreased migration (P < 0.001) and invasion (P < 0.001). Compared with controls, EphB4 siRNA–1,2-dioleoyl-sn-glycero-3-phosphatidylcholine alone significantly reduced tumor growth in the A2780-cp20 (48%, P < 0.05) and IGROV-af1 (61%, P < 0.05) models. Combination therapy with EphB4 siRNA–1,2-dioleoyl-sn-glycero-3-phosphatidylcholine and docetaxel resulted in the greatest reduction in tumor weight in both A2780-cp20 and ...
JNCI: Journal of the National Cancer Institute, 2009
Ovarian cancer is the most common cause of death from a gynecologic malignancy (1). Although most... more Ovarian cancer is the most common cause of death from a gynecologic malignancy (1). Although most patients with advanced-stage ovarian cancer will die of the disease, more than 70% have a favorable initial response to surgery and chemotherapy and a substantial fraction will respond to second-line therapies (2 , 3). Systemic chemotherapy is widely used but is frequently associated with intolerable side effects (4). Given the high mortality rate of ovarian cancer, new therapies are urgently needed to target the tumor while sparing normal tissues.
Journal of Clinical Investigation, 2010
Chronic stress is associated with hormonal changes that are known to affect multiple systems, inc... more Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAK Y397 , which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAK Y397 , which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management. Conflict of interest: The authors have declared that no conflict of interest exists.
Journal of Biological Chemistry, 2010
A growing number of studies indicate that chronic stress can accelerate tumor growth due to susta... more A growing number of studies indicate that chronic stress can accelerate tumor growth due to sustained sympathetic nervous system activation. Our recent findings suggest that chronic stress is associated with increased IL8 levels. Here, we examined the molecular and biological significance of IL8 in stress-induced tumor growth. Norepinephrine (NE) treatment of ovarian cancer cells resulted in a 250-300% increase in IL8 protein and 240-320% increase in its mRNA levels. Epinephrine treatment resulted in similar increases. Moreover, NE treatment resulted in a 3.5-4-fold increase in IL8 promoter activity. These effects were blocked by propranolol. Promoter deletion analyses suggested that AP1 transcription factors might mediate catecholamine-stimulated up-regulation of IL8. siRNA inhibition studies identified FosB as the pivotal component responsible for IL8 regulation by NE. In vivo chronic stress resulted in increased tumor growth (by 221 and 235%; p < 0.01) in orthotopic xenograft models involving SKOV3ip1 and HeyA8 ovarian carcinoma cells. This enhanced tumor growth was completely blocked by IL8 or FosB gene silencing using 1,2-dioleoyl-snglycero-3-phosphatidylcholine nanoliposomes. IL8 and FosB silencing reduced microvessel density (based on CD31 staining) by 2.5-and 3.5-fold, respectively (p < 0.001). Our findings indicate that neurobehavioral stress leads to FosB-driven increases in IL8, which is associated with increased tumor growth and metastases. These findings may have implications for ovarian cancer management.
International Journal of Infectious Diseases, 2008
e341 were 46 (28.2%) aboriginals, 3 (1.8%) incarcerated, and 3 (1.8%) living in a long term care ... more e341 were 46 (28.2%) aboriginals, 3 (1.8%) incarcerated, and 3 (1.8%) living in a long term care facility. Forty-one (24.0%) were new patients and 83 (48.5%) had treatment failure or relapse. Exclude the new patients of our MDR program, 25 (19.8%) developed both isoniazid and rifampin resistance within one year of starting anti-tuberculosis treatment, and 81 (64.3%) developed resistance after more than two years of treatment. Drug resistance was highest against fluoroquinolone (49%). Conclusion: Having prior treatment with poor compliance was considered the major determinant of developing MDR-TB. However, 24% of our MDR-TB patients were new patients, indicating possible transmission of MDR-TB in Taiwan. Aggressive TB control efforts are needed to prevent further development and spread of MDR-TB.
Gynecologic Oncology, 2011
The overall survival rate of patients with epithelial ovarian cancer (EOC) is poor and most of th... more The overall survival rate of patients with epithelial ovarian cancer (EOC) is poor and most of the patients who are alive at five years have active disease. Thus, 10-year survival may be a more appropriate endpoint. By definition, relative survival (RS) adjusts for the general survival rate of the population for that race, sex, age and date at which the diagnosis was coded. The 10-year RS for ovarian cancer using SEER data has previously been reported, but was not limited to epithelial histologies. Our objective was to measure RS in EOC over 10 years, stratified by stage, race, age, classification of residence and surgery as the first course of treatment. Using the SEER 1995-2007 database, cases were identified using the International Classification of Diseases codes for EOC. Inclusion criteria included malignant behavior, first primary, actively followed, and age ≥20 years. Exclusion criteria included death certificate-or autopsy-only cases. Using the actuarial life table method, RS over 10 years was calculated, stratified by stage, classification of residence, surgery as the first course of treatment, race and age. Results: Forty thousand six hundred ninety-two patients met inclusion criteria. Stage distribution was 20, eight, 39, 27, and 7% for stages I, II, III, and IV, and unknown, respectively. The first course of treatment was surgery in 78%. Overall RS was 65, 44, and 36% at two, five and 10 years, respectively. The slope of decline in RS was reduced for years five-10 as compared with years one-five after diagnosis. RS at five years was 89, 70, 36, and 17% for stages I, II, III, and IV, respectively. RS at 10 years was 84, 59, 23, and 8% for stages I, II, III, and IV, respectively. RS was comparable for patients living in rural and urban settings. At all stages, patients who underwent surgery as their first course of treatment had better RS than those who did not have surgery. When RS was compared between races, blacks had the poorest survival. At all stages, advanced age at time of diagnosis was associated with decreased RS. Conclusions: Although advanced EOC is associated with a poor prognosis, updated data demonstrate that survival rates adjusted for survival in the general population (RS) at five years are improved over historic reports and the 10-year RS for stage III is higher than expected. These data provide the physician and the patient with more accurate prognostic information, particularly for advanced-stage disease.
Drug Resistance Updates, 2009
Platinum based drugs continue to be the mainstay of therapy for ovarian cancer. Along with advers... more Platinum based drugs continue to be the mainstay of therapy for ovarian cancer. Along with adverse effects, chemoresistance (intrinsic or acquired) has become a major limitation in the management of recurrent disease. Even though much is known about the effects of platinum drugs on cancer cells, the mechanisms underlying resistance are poorly understood. In this review, we summarize the current data on chemoresistance and discuss novel strategies to reverse resistance to platinum-based drugs. The most important targets highlighted here include Aurora kinases, PARP, ATP7B, and ERCC1. Furthermore, we discuss the implications of these novel approaches for ovarian cancer treatment.
Clinical Cancer Research, 2010
Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (... more Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle (RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA). Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometry and fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovarian carcinoma. Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of multiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKOV3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP). Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targeted siRNA into the ανβ3 integrin...
Clinical Cancer Research, 2011
Purpose: Src is an attractive target because it is overexpressed in a number of malignancies, inc... more Purpose: Src is an attractive target because it is overexpressed in a number of malignancies, including ovarian cancer. However, the effect of Src silencing on other Src family kinases (SFKs) is not known. We hypothesized that other SFK members could compensate for the lack of Src activity. Experimental Design: Cell viability after either Src or Fgr silencing was examined in ovarian cancer cell lines by MTT assay. Expression of SFKs after Src silencing in ovarian cancer cells was examined by real-time reverse transcriptase (RT)-PCR. Therapeutic effect of in vivo Src and/or Fgr silencing was examined using siRNA incorporated into chitosan nanoparticles (siRNA/CH-NP). Microvessel density, cell proliferation, and apoptosis markers were determined by immunohistochemical staining in ovarian tumor tissues. Results: Src silencing enhanced cytotoxicity of docetaxel in both SKOV3ip1 and HeyA8 cells. In addition, Src silencing using siRNA/CH-NP in combination with docetaxel resulted in signif...
Clinical Cancer Research, 2009
Purpose: Surgical stress has been suggested to facilitate the growth of preexisting micrometastas... more Purpose: Surgical stress has been suggested to facilitate the growth of preexisting micrometastases as well as small residual tumor postoperatively. The purpose of this study was to examine the effects of surgical stress on ovarian cancer growth and to determine underlying mechanisms responsible for increased growth. Experimental Design: To mimic the effects of surgery, we did a laparotomy or mastectomy under isoflurane inhalation on athymic nude mice 4 days after i.p. tumor cell injection. Propranolol infusion via Alzet pumps was used to block the influence of sympathetic nervous system activation by surgical stress. Results: In both HeyA8 and SKOV3ip1 models, the mice in the laparotomy and mastectomy groups had significantly greater tumor weight (P < 0.05) and nodules (P < 0.05) compared with anesthesia only controls. There was no increase in tumor weight following surgery in the β-adrenergic receptor–negative RMG-II model. Propranolol completely blocked the effects of surgi...
Clinical Cancer Research, 2011
Purpose: To study the role of survivin and its splice variants in taxane-resistant ovarian cancer... more Purpose: To study the role of survivin and its splice variants in taxane-resistant ovarian cancer. Experimental Design: We assessed the mRNA levels of survivin splice variants in ovarian cancer cell lines and ovarian tumor samples. siRNAs targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA) in vitro and orthotopic murine models of ovarian cancer. The mechanism of cell death was studied in taxane-resistant ovarian cancer cells and in tumor sections obtained from different mouse tumors. Results: Taxane-resistant ovarian cancer cells express higher survivin mRNA levels than their taxane-sensitive counterparts. Survivin 2B expression was significantly higher in taxane-resistant compared with -sensitive cells. Silencing survivin 2B induced growth inhibitory effects similar to silencing total survivin in vitro. In addition, survivin 2B-siRNA incorporated into DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) nanoliposomes resulted in s...
Clinical Cancer Research, 2011
Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor gr... more Purpose: Increased adrenergic activity in response to chronic stress is known to promote tumor growth by stimulating the tumor microenvironment. The focus of the current study was to determine whether dopamine, an inhibitory catecholamine, could block the effects of chronic stress on tumor growth. Experimental Design: Expression of dopamine receptors (DR1–DR5) was analyzed by reverse transcriptase-PCR and by Western blotting. In vitro effects of dopamine on cell viability, apoptosis, and migration were examined. For in vivo therapy, murine and human DR2-siRNAs were incorporated into chitosan nanoparticles (CH-NP). Results: In this model of chronic stress, tumoral norepinephrine levels remained elevated whereas dopamine levels were significantly decreased compared with nonstressed animals. Daily restraint stress resulted in significantly increased tumor growth in both immunodeficient (SKOV3ip1 and HeyA8) and immunocompetent (ID8) ovarian cancer models. This increase was completely bl...
Clinical Cancer Research, 2009
Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. ... more Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant ce...
Cancer Research, 2011
Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important ... more Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with...
Cancer Research, 2010
RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of p... more RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compar...
Cancer Letters, 2013
The present study was undertaken to determine the expression and biological significance of HORMA... more The present study was undertaken to determine the expression and biological significance of HORMAD1 in human epithelial ovarian carcinoma. We found that a substantial proportion of human epithelial ovarian cancers expressed HORMAD1. In vitro, HORMAD1 siRNA enhanced docetaxel induced apoptosis and substantially reduced the invasive and migratory potential of ovarian cancer cells (2774). In vivo, HORMAD1 siRNA-DOPC treatment resulted in reduced tumor weight, which was further enhanced in combination with cisplatin. HORMAD1 gene silencing resulted in significantly reduced VEGF protein levels and microvessel density compared to controls. Our data suggest that HOMRAD1 may be an important therapeutic target.
Uploads
Papers by Mian Nasir Shahzad