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PDF file - 126K, MNP-CUR nanoparticles efficiently internalize in cancer cells.

Supplementary Figure S4. Schematic representation of the treatment regime of xenograft mice.

The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer. Cancer Res; 75(11); 2292–304. ©2015 AACR.

Legends for Supplementary Figures S1-S4.

Background: Hepatocellular carcinoma (HCC) is a lethal cancer with a dismal 5-year survival rate as the standard therapy available Sorafenib (SRB), is only effective in extending survival for a subset of patients. Resistance to apoptosis is a defining characteristic of cancer cells and a crucial factor in cancer recurrence and relapse. Although, anticancer potential of the FDA-approved compound Ormeloxifene (ORM), which has well-defined pharmacokinetic and pharmacodynamic properties, has been explored in a variety of cancers, its efficacy in HCC has remained unknown. Therefore, the purpose of this study was to investigate the role and molecular mechanisms underlying ORM in SRB-induced apoptosis in HCC. Methods: In this study, we investigated the efficacy of ORM in hepatocellular carcinoma cell (HepG2, Hep3B, C3a, and SKHep-1) lines alone, and in combination with SRB. Cell proliferation was determined by MTT and xCELLigence assay. Effect of ORM on clonogenic potential of HCC cells was examined by colony formation assay. Effect of ORM on apoptosis induction was performed by Annexin V assays. The cell migration assay was performed using Boyden chamber and cell invasion assay was done by Matrigel invasion chambers. Following treatment in hepatocellular carcinoma cells, Western blotting, quantitative polymerase chain reaction (qPCR), and immunofluorescence studies were carried out in order to investigate the effects of ORM, SRB alone, and/or combination on pro-apoptotic, anti-apoptotic, EMT, and associated signaling effector proteins. Results: By employing MTT and xCELLigence assays, our study showed that ORM induces dose- and time-dependent inhibition of cell proliferation in hepatocellular cancer (HepG2, Hep3B, C3a, and SKHep-1) cells at various concentrations (0, 5, 10, and 15 µM). The combination of ORM and SRB together had a significant synergistic inhibitory effect on HCC cells. Additionally, compared to ORM and SRB treatment alone, Annexin V staining showed that combined treatment of ORM and SRB significantly enhance the apoptosis induction in hepatocellular carcinoma cells. Mechanistically, the expression of anti-apoptotic markers like Mcl-1, Bcl-2, and Bcl-xl is significantly reduced when ORM and SRB are combined as evidenced by Western blotting. Similarly, the inhibition of colony formation, invasion, and migration of hepatocellular carcinoma cells following co-treatment was significantly higher as compared to either treatment alone. In addition, the expression of EMT markers like N-cadherin, snail, vimentin, and MMPs in HCC cells is down-regulated following combined treatment compared to ORM and SRB alone. Conclusion: Taken together, ORM displayed an effective chemo-therapeutic and chemo-sensitizing agents in treatment of hepatocellular carcinoma. Citation Format: Mohammed Sikander, Shabnam Malik, Anyssa Rodriguez, Daniel Zubieta, Sheema Khan, Fathi T. Halaweish, Murali M. Yallapu, Subhash C. Chauhan, Meena Jaggi. Molecular mechanism of ormeloxifene mediated chemo-sensitivity in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 399.

Background: As per key statistics of American Cancer Society 2021, Pancreatic Cancer (PanCa) affects around 60,430 persons (31,950 men and 28,480 women) a year in the U.S. and is tricky to diagnose and treat. PanCa is the 4th leading cause of cancer death with a 5-year survival rate of only 9%, along with a poor prognosis. Generally (around 85-90%) PanCa are adenocarcinomas, such as Pancreatic ductal adenocarcinomas (PDAC). PDAC is now one of the most challenging tumor entities worldwide, characterized as a highly aggressive disease with dismal overall prognosis with a mortality rate near the incidence rate. For PDAC, no early tumor specific biomarker is available, therefore, novel early detection biomarker strategy is immediately required to upgrade the narrow diagnostic portfolio against PanCa. Considering this alarming situation, our team has identified a novel oncogenic protein, Carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7). Our studies suggested high CEACAM7 expression in PDAC tumors and its association with patient survival. In this study we have investigated the potential role of CECAM7 for early PDAC diagnosis and its role in PDAC progression. Methodology: This study includes bioinformatics pre-screening using publicly available cancer databases followed by molecular biology techniques. To detect the degree of expression of CEACAM7 in normal pancreatic cell (HPNE) and PDAC progressive cellular models, we have minutely scrutinized the PDAC cell panel (HPAF-2, SU86.86 & Panc-1) & TMAs of PDAC patients, in terms of mRNA & protein expression level of CEACAM7. The confocal microscopy was performed to identify the intensity and localization of CEACAM7 protein. IHC analysis was also performed to identify the protein expressions in the pancreatitis and tumor cores along with their locations, grading and Mean Composite Score. Results: Bioinformatic results confirmed the relevance of CEACAM7 as a potential early diagnostic and prognostic marker of PDAC. HPAF2 (well differentiated) expressed highest mRNA and protein expression analyses, followed by SU86.86 (moderately differentiated) and very low expression in AsPc1 (poorly differentiated), and Panc1 (poorly differentiated). The IHC analyses of TMAs exhibited negligible or faint staining in pancreatitis (chronic and acute) and most of the positive cases were in tumor grading 1 & 2. Conclusion: Our observations clearly cite that CEACAM7 can serve as a potential early diagnostic and/or prognostic marker of PDAC and may also potentiate the sensitivity of the existing biomarker panel of PDAC. However, further studies are warranted to determine its clinical significance. Keywords: Pancreatic cancer, PDAC, CEACAM7, Early detection biomarker, Tumor grading Citation Format: Anupam Dhasmana, Swati Dhasmana, Sheema Khan, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. CEACAM7 an early detection biomarker for pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4314.

Aim: To compare and correlate the serum insulin and lipoprotein levels in diabetics and non-diabetics. Methods: A cross-sectional study with total of 88 subjects. These were divided into three groups i.e. A, B, C. Group A constitutes 34 cases of newly diagnosed T2DM and group B includes 34 cases on oral hypoglycemic treatment. Group C includes 20 subjects as controls which were age and sex matched. Blood glucose level, HDL-c and LDL-c were done in the lab. By usual methods and insulin level was done by ELISA methods. Results: In diabetic groups i.e. A and B, mean insulin levels were significantly lower and glucose and LDL-c levels were significantly higher than control group i.e. group C. HDL-c level was similar in diabetic groups (A and B) and control group (group C). There was found no correlation between insulin and lipoproteins in this study. Conclusions: Insulin level was higher in control group as compared to study subjects and this difference was not statistically significant. LDL-c level was higher in diabetic group i.e. A and B as compared to control group (C) and this difference was statistically significant. Association between insulin and lipoproteins is non significant statistically. Keywords: Insulin, Lipoprotein, Type-2 diabetes

Aim: To find out the serum adiponectin levels in patients of type 2 diabetes and non diabetics. Methods: A cross-sectional, comparative study included 88 subjects. Out of 88 cases, 68 were diagnosed cases of T2DM. In 68 diagnosed cases, 34 were newly diagnosed cases of T2DM without any treatment and 34 were on oral hypoglycemic medicine. 20 subjects were controls which were age and sex matched. Serum adiponectin levels were estimated by using ELISA procedure. Total cases were divided into Group A: 34 newly diagnosed patients of T2DM without any treatment. Group B: 34 patients of T2DM on oral hypoglycemic medicine and Group C: 20 subjects of normal controls. Results: In our study, mean  SD values of adiponectin levels in non-diabetics in Group C were 1560 1044.6 ng/ml. In group A, mean  SD values of adiponectin levels were 1153±792.6ng/ml and in group B, mean  SD values of adiponectin levels were 1601±1210.5ng/ml. The adiponectin levels were lower in diabetics without medication (A) as compared to the control group (C). The difference was statistically significant between groups A vs B and A vs C while the difference was statistically non significant between groups B vs C. Conclusion: The adiponectin levels were lower in diabetics without medication (group A) as compared to the diabetics with drugs (group B) and control group (C). The difference was statistically significant between groups A vs B and A vs C while the difference was statistically insignificant between groups B vs C. Keywords: Adiponectin level, Lipids, Type-2 diabetes mellitus

Gastrointestinal (GI) cancers are one of the leading causes of death worldwide. Although various approaches are implemented to improve the health condition of GI patients, none of the treatment protocols promise for eradicating cancer. However, a treatment mechanism against any kind of disease condition is already existing executing inside the human body. The 'immune system' is highly efficient to detect and destroy the unfavourable events of the body including tumor cells. The immune system can restrict the growth and proliferation of cancer. Cancer cells behave much smarter and adopt new mechanisms for hiding from the immune cells. Thus, cancer immunotherapy might play a decisive role to train the immune system against cancer. In this review, we have discussed the immunotherapy permitted for the treatment of GI cancers. We have discussed various methods and mechanisms, periodic development of cancer immunotherapies, approved biologicals, completed and ongoing clinical trials, role of various biopharmaceuticals, and epigenetic factors involved in GI cancer immunotherapies (graphical abstract Figure 1).

The present research was carried out in district Ganderbal of Jammu and Kashmir to study the entrepreneurial behavior of grape growers. . By using proportionate allocation method, a sample of 120 grape growers was taken for collecting the primary data with the aid of an interview schedule. Data derived from the interviewees of the sampled growers was examined using competent statistical procedures. Most (73%) of the respondents showed medium, high (15%) and low (12%) entrepreneurial behavior. In order of ranking the constraints were reported as, most 85% of the growers reported that no bowers were provided to them’, 75.83% percent of the growers indicated the small fruit size of the berries’, 61.66% reported the irregular rains’, 35% indicated onset of diseases’, 29.16% indicated fluctuation of market prices, 19.16% reported distant markets’ and 15% indicated no net availability’.

The study was carried out during 2018-2019 to know the socio-economic status of the chilli growers, marketing of chilli and constraints faced by the chilli growers in district Baramulla. A sample size of 50 chilli growers was selected randomly from 5 villages of agricultural zone Nehalpora of district Baramulla. The study reveals that majority (60%) of the respondents were in the age group (35-53 yrs). It was found that 30 per cent of the respondents were illiterate. About 45% of the respondents were having farming as the main occupation. As far as annual income is concerned, major source of income of the growers was from agriculture (72 lakhs), followed by horticulture (39.70 lakhs) and vegetables (3.23 lakhs). About 45% of the area was irrigated in the village, 40.87% under orchards, 9.48% under chilli. Majority of the growers used television (74%) regularly as a source of mass media followed by other means of media like internet (30%). Majority (84%) of the growers had occasional extension contacts. About 56 per cent of the respondents were having medium level of scientific orientation followed by low (30%) and high (14%). As far as the marketing of the chilli is concerned, majority of the growers (56%) sold their crop in local market while only (44%) of the growers sold their crop at a distant market. The reason might be that the growers preferred their local market rather than going for distant places because of the less time available. The major resource constraints in chilli production technology as mentioned by the respondents were unavailability of improved seeds of vegetables (90%) high cost of pesticides (70%), scattered and small size land holdings (60%) and lack of irrigation facilities (40%) Poor marketing facilities (80%), distantly located markets (30%), bad condition of roads (90%), non-remunerative prices (84%) and lack of transportation facilities and high transportation charges (18%) were the major marketing constraints. 90 per cent of the farmers had a constraint of Lack of training on scientific vegetable production technology and Non-availability of facilities of soil testing (80% ), while as 40 per cent had mentioned Lack of knowledge about improved varieties, seed rate and sowing time as other constraint faced.

Objectives: To determine the frequency and severity of depression and anxiety in patients who had undergone total abdominal hysterectomy with bilateral salpingo-oophorectomy. Study Design: A cross-sectional study. Setting: Department of Obstetrics and Gynecology, Isra University Hospital, Hyderabad, Sindh. Period: From 14th January 2015 to 14th January 2016. Material & Methods: Total 136 women were included in the study that had undergone total abdominal hysterectomy along with bilateral salpingo-oophorectomy. Women who had an obstetrical hysterectomy, vaginal hysterectomy, subtotal hysterectomy, hysterectomized women with pre-existing depression and psychosis and women who did not agree to participate were excluded. Anxiety and depression were labelled along with severity as per operational definition using Zung self-rating depression and Zung self-rating anxiety scale and were analyzed by using SPSS 11.0. Result: Majority of the women were married (88.2%), educated (66.9%), and grand multiparous (46.3%). Commonest age group was more than 40 years (29.4%). Anxiety was present in 27.2% whereas depression was seen in 32.4% women. Conclusion: There was a highly significant association of age, parity and level of education with anxiety as well as depression. Majority of the women suffered from mild to moderate level of anxiety and depression.

Objective: To compare the bimanual hand function in children with and without mirror movements and to find the correlation between the bimanual hand function, functional independence and severity of mirror movements. Design: Comparative cross sectional study. Place and duration of the study: The study was conducted in special education schools of Lahore and Islamabad, Pakistan, from August 2017 to January 2018. Patients and Methods: Children suffering from spastic cerebral palsy including both males and females in age groups ranging from 5 to 18 years who were able to make a gross grip were included in the study. Children having low tone, not able to make a gross grip and with any deformity that makes the assessment difficult were excluded. The non probability sampling technique was used. The Woods and Teuber criteria were used to check the severity of the mirror movements and bimanual activities to compare the hand functions. Data was analyzed using SPSS 21. Results: The mean age of the participants was 11.17±3.69 years. Children who presented with mirror movements had a mean age of 12±3.70 years and those without mirror movements had mean age of 10.72±3.63 years. According to the results of the bimanual task scale, there was no difference in the bimanual hand function of children with mirror movements and without mirror movements (P value >0.05) Conclusion: According to the results of the bimanual task scale there is no significant difference in the bimanual hand function of children suffering from spastic cerebral palsy, either with with or without mirror movements. However, functional independence was found more in the children suffering from cerebral palsy who presented without mirror movements.

Pancreatic cancer has the worst prognosis and lowest survival rate among all cancers. Pancreatic cancer cells are highly metabolically active and typically reprogrammed for aberrant glucose metabolism; thus they respond poorly to therapeutic modalities. It is highly imperative to understand mechanisms that are responsible for high glucose metabolism and identify natural/synthetic agents that can repress glucose metabolic machinery in pancreatic cancer cells, to improve the therapeutic outcomes/management of pancreatic cancer patients. We have identified a glycoside, steviol that effectively represses glucose consumption in pancreatic cancer cells via the inhibition of the translation initiation machinery of the molecular components. Herein, we report that steviol effectively inhibits the glucose uptake and lactate production in pancreatic cancer cells (AsPC1 and HPAF-II). The growth, colonization, and invasion characteristics of pancreatic cancer cells were also determined by in vitro functional assay. Steviol treatment also inhibited the tumorigenic and metastatic potential of human pancreatic cancer cells by inducing apoptosis and cell cycle arrest in the G1/M phase. The metabolic shift by steviol was mediated through the repression of the phosphorylation of mTOR and translation initiation proteins (4E-BP1, eIF4e, eIF4B, and eIF4G). Overall, the results of this study suggest that steviol can effectively suppress the glucose metabolism and translation initiation in pancreatic cancer cells to mitigate their aggressiveness. This study might help in the design of newer combination therapeutic strategies for pancreatic cancer treatment.

Aim: To see the frequency of fetal haemoglobin level in siblings of beta thalassemia major (BTM) patients. Study population: A total of 400 subjects were included. Grouping: Group A includes normal siblings of BTM cases, B includes beta thalassemia trait (BTT) siblings of BTM cases and group C were healthy controls. Selection criteria: Asymptomatic siblings of diagnosed cases of beta thalassemia major were included. Patients with history or diagnosis of any acute or chronic illnesses were excluded from the study. 5 ml of blood was taken in EDTA vial and used for haemoglobin electrophoresis. Results: In 200 cases, 78% (156) were beta thalassemia trait (BTT) siblings and fetal Hb was also more in BTT siblings i.e. Group B. Conclusion: Fetal haemoglobin (HbF) was increased in BTT siblings (group B) of BTM cases. Keywords: Hb electrophoresis, fetal Hb, beta thalassemia major

Aim: Prevalence and antimicrobial susceptibility pattern of gram negative rods in blood cultures. Methods: Cross-sectional study performed in Pathology section, Pak Red Crescent Teaching hospital, Lahore for one year i.e. 01-11-2019 to 31-12-2020. After approval from ethical committee, 1100 blood samples were taken for C/S from various sections of the Hospital. The antimicrobial sensitivity was done by Kirby-Bauer disc diffusion method as approved by Clinical and Laboratory Standard Institute (CLSI). Results: Out of 1100 Blood samples, 65 samples of blood cultures were positive for Gram –ve Rods. The prevalence of Gram negative Rods in blood specimens was only 6%. Out of 6% gram –ve rods, 12% were E.coli, 9% Klebsiella, 3% Enterobacter cloacae, 45% Salmonella, 2% Proteus, 17% Pseudomonas, 10% Acinetobacter and 2% were serretia. Conclusion: The prevalence of Gram negative rods in blood specimens was only 6%. Keywords: Gram negative rods, blood cultures, Antibiotic sensitivity pattern

Exosomes are nanoscale vesicles generated by cells for intercellular communication. Due to their composition, significant research has been conducted to transform these particles into specific delivery systems for various disease states. In this review, we discuss the common isolation and loading methods of exosomes, some of the major roles of exosomes in the tumor microenvironment, as well as discuss recent applications of exosomes as drug delivery vessels and the resulting clinical implications.

Paclitaxel (PTX) is a gold standard chemotherapeutic agent for breast, ovarian, pancreatic and non-small cell lung carcinoma. However, in clinical use PTX can have adverse side effects or inadequate pharmacodynamic parameters, limiting its use. Nanotechnology is often employed to reduce the therapeutic dosage required for effective therapy, while also minimizing the systemic side effects of chemotherapy drugs. However, there is no nano formulation of paclitaxel with chemosensitization motifs built in. With this objective, we screened eleven pharmaceutical excipients to develop an alternative paclitaxel nanoformulation using a self-assembly method. Based on the screening results, we observed tannic acid possesses unique properties to produce a paclitaxel nanoformulation, i.e., tannic acid-paclitaxel (TAP). This stable TAP nanoformulation, referred to as TAP nanoparticles (TAP NPs), showed a spherical shape of 102.22±14.05 nm and negative zeta potential of −8.85±0.44 mV. The presence of PTX in TAP NPs was confirmed by Fourier Transform Infrared (FTIR) spectra, thermogravimetric analyzer (TGA) scans, and X-ray diffraction (XRD). Encapsulation efficiency of PTX in TAP NPs was determined to be > 96.49±0.43 %. Intracellular drug uptake of plain drug PTX on breast cancer cells (MDA-MB-231) shows more or less constant drug concentration from 2–6 hours, suggesting drug efflux by the P-gp transporters, over TAP NPs, in which PTX uptake was more than 95.52+11.01% in 6 hours, as analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Various biological assays such as proliferation, clonogenic formation, invasion, and migration confirm superior anticancer effects of TAP NPs over plain PTX at all tested concentrations. P-gp expression Rhodamine-123 (RH123), beta-tubulin stabilization, Western blot, and microarray analysis further confirm the improved therapeutic potential of TAP NPs. These results suggest that the TAP nanoformulation provides an important reference for developing a therapeutic nanoformulation affording pronounced, enhanced effects in breast cancer therapy.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. About 90% of all cancer-related deaths are due to the development of metastatic sites in the body. 40-50% of colorectal cancer patients develop metastasis at some point during their fight with the disease. Understanding the mechanism of metastasis in colorectal cancer is vital. Metastasis is a multistep process; anchorage-independent survival after intravasation of cells from the primary tumor site is a crucial step. In our lab, we have previously demonstrated that MUC13 plays an essential role in the CRC progression by modulating anti-apoptotic pathways and survival proteins expression. Under anchorage-independent growth conditions, we have identified that MUC13, in correspondence with YAP1, is the key upregulated protein and is responsible for increased survival. Isogenic CRC cell lines SW480 (non-metastatic) and SW620 (metastatic) on low adherence growth conditions are applied in these studies. In addition, lentiviral transduced stable overexpression and knockdown cell lines were generated for MUC13 and YAP1 mechanistic studies. The overexpression of MUC13 in non-metastatic SW480 cells (low MUC13 expressing) increased anchorage-independent survival and enhanced tumorigenesis compared to SW480+Vector cells, contrary results were found upon MUC13 knockdown in SW620 cells (high MUC13 expressing). In vitro results were recapitulated in the in vivo mouse model system and human CRC tissues. In Proximity Ligation Assays (PLA), we found an increased nuclear localization of the survival complex YAP1/β-catenin in MUC13 overexpressing cells. In contrast, MUC13 knockdown resulted to the lower aboudance of survival complex in the nucleus. Immunoprecipitation validates the protein-protein interaction between MUC13 and YAP1. YAP1 knockdown in MUC13 overexpressing cells showed a decrease in survival, indicating the necessary functional complex formation between MUC13 and YAP1. MUC13 and YAP1 expression in human CRC tissue were highest at Stage II. However, YAP1 expression increased when MUC13 was observed in the nucleus. This supports the notion that MUC13 is critical in enhancing CRC metastasis through molecular interaction with YAP1. For the first time, this study demonstrates complex formation between MUC13 and YAP1 and defines their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Manish K. Tripathi, Radhika Sekhri, Sudhir Kotnala, Bilal Hafeez, Sheema Khan, Nadeem Zafar, Murali Yallapu, Meena Jaggi, Subhash Chauhan. MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 992.

Introduction: In pancreatic ductal adenocarcinoma (PDAC), low patient survival rate remains a problem. The activating point mutation of KRAS on codon-12 is present in 70-95% of PDAC cases and so far, no success has been achieved to inhibit KRAS. KRASG12D regulates cell proliferation, differentiation, apoptosis; recent preliminary and published studies show high Galectin-1 (Gal-1) levels in both PDAC and stromal cells, which modulate tumor microenvironment and metastasis. Therefore, we have developed a novel combination therapy for PDAC by targeting mutated KRASG12D and Gal-1 to target both proliferation and metastasis in PDAC. This includes the delivery of KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: Our patented SPION nano-formulation was used to deliver siKRASG12D and investigated in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a mouse model. Results: Our results demonstrate optimal particle size/zeta potential of SP-siKRAS formulation. SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation. It inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells. This resulted in activation of death related mechanisms, such as Bax, bcl-2, PARP cleavage in KRASG12D cells. Interestingly, the formulation was highly effective in inhibiting KRASG12D and growth of tumor spheroid in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. This further provides a clinical validation demonstrating potential of SP-siKRAS particles to efficiently silence KRAS expression. SP-siKRAS also exhibited hemocompatibility and stability suggesting its potential of silencing KRAS without being toxic to the body. The formulation efficiently exhibited KRasG12D silencing and inhibited tumor growth and metastasis in nude mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Martinez Bulnes, Poornima Shaji, Nirnoy Dan, Melida Cantu, Shabnam Malik, Stephen Behrman, Swathi Holla, Murali Mohan Yallapu, Meena Jaggi, Subhash C. Chauhan, Sheema Khan. Novel therapy targeting mutant-KRASG12D and galectin-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1038.

Background: Pancreatic cancer remains 3rd deadliest disease, with less than 7-10% survival rate. Little progress has been seen in patient’s outcome due to high desmoplasia and chemo-resistance. Immunotherapy has shown promising results in cancers, except pancreatic cancer due to their characteristic fibrotic tumor microenvironment. The therapies are unable to penetrate fibrotic tumor leading to insufficient availability of therapeutic drugs at the tumor site. A recently identified mucin, MUC13 is aberrantly expressed in pancreatic tumors but not in normal pancreas, that makes it an excellent protein tumor target. This study is unique as it utilizes MUC13Ab for targeting the pancreatic tumor site and SPION nanoparticle system for delivering the stroma depleting drug (curcumin), which would help in improving immunotherapy response. Methods: The inhouse generated MUC13Ab have been conjugated with our recently developed novel patented superparamagnetic iron oxide nanoparticles (SPIONS). Conjugation efficiency of the SPION-Anti-MUC13 particles was seen through cell uptake studies, by measuring fluorescence intensity, Prussian blue staining. Invasion assay and migration assay was carried out on KPC cells. We have used female C57BL/6J black mice, orthotopic mice model for investigating targeting efficacy of MUC13-SPION-CUR. Immune checkpoint therapy (PDL-1 and CTLA-4) was administrated along with MUC13-SPION-CUR and conjugated with fluorescent indocyanine green (ICG) dye for monitoring the tumor growth. Further, immunostimulatory effect of the nano formulation was done using flow cytometry. Results: Our results showed that MUC13Ab conjugated SPIONS can efficiently internalize the PDAC cells. SPION-MUC13 using Indocyanine dye (ICG) specifically reached to the tumor site in an orthotopic syngeneic mouse model of PDAC as indicated by ICG fluorescence. Additionally, the combination formulation inhibited the tumor growth and showed more survival rate with CTLA-4. The combined treatment with CTLA-4 increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 43% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 23.8% (FoxP3+CD25+CD45+CD3+) in KrasG12D; LSL-Trp53R172H syngeneic mouse model of PDAC. Similar results were observed in SP-CUR-M13+PDL-1 group, which showed reduction in MDSCs (by 26.6%) and Tregs (by 0.1%) as compared with PDL-1 alone. Conclusion: The formulation softens up the tumors for therapies that resulted in improved response to checkpoint immunotherapies in a pancreatic orthotopic mice model. Therefore, this study indicates high significance of MUC13-SPIONS-CUR for achieving pancreatic tumor specific delivery of drugs. This study has a potential to reduce morbidity and mortality caused by the disease and improve survival in patients. Citation Format: Poornima Shaji, Ana Martinez, Melida Flores Cantu, Anupam Dhasmana, Neeraj Chauhan, Fnu Shabnam, Vincent Diego, Meena Jaggi, Stephen Behrman, Murali M. Yallapu, Subhash C. Chauhan, Sheema Khan. A novel approach to target tumor immune microenvironment and improve checkpoint immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB007.

Background: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer with approximately 41,000 new diagnosis and 29,000 fatalities every year only in the U.S. Severe toxicity and poor response to available chemotherapeutic agents make HCC clinical management extremely difficult. Aberrant β-catenin signaling is found to play a critical role in differentiation grades of HCC patients. Thus, attempts to find more potent and non-toxic β-Catenin inhibitors is urgently needed for cancer therapy. Notably, ormeloxifene (ORM), clinically approved selective estrogen receptor modulator with therapeutic index, has shown excellent anticancer activity, rendering it as an ideal candidate for repurposing. In this study, we investigated the anti-cancer potential of ORM against hepatocellular carcinoma. Methodology: Cell proliferation and colony formation assays were performed to assess the therapeutic activity of ORM in human hepatocellular carcinoma (HepG2, SK-HEP-1, Hep3B, and C3A) cells. Boyden chamber and Matrigel assays were carried out for investigating the effect of ORM on migration and invasion abilities of HCC cells, respectively. The effects of ORM on β-catenin and EMT associated proteins were analyzed through Western blotting and qPCR. Confocal microscopy was used to determine the β-catenin nuclear localization following ORM treatment in HCC cells. Results: As compared to vehicle-treated group, ORM treatment (2.5-20 µM) suppressed proliferation and colony formation in human hepatocellular carcinoma cells in a dose and time-dependent manner. Moreover, ORM treatment suppresses the migration and invasion of human hepatocellular carcinoma cells as shown by wound healing and Matrigel invasion assay, respectively. ORM effectively inhibited the protein levels and mRNA expression of total β-catenin. Additionally, our confocal microscopy results further showed reduced nuclear translocation of β-catenin following the ORM treatment. ORM treatment inhibited epithelial-to-mesenchymal transition (EMT) process as evident by repression of N-cadherin, Slug, Snail, vimentin, MMPs (MMP2 and MMP3), and induced the expression of pGSK3β. Experiments are also being conducted to see how ORM affects epigenetic markers linked to EMT and β-catenin signaling. Conclusion: Taken together, ORM inhibited β-catenin signaling and exhibited potent anticancer effects against HCC and could be investigated further as a novel therapeutic modality for HCC treatment. Citation Format: Mohammed Sikander, Shabnam Malik, Anyssa Rodriguez, Molly Vela, Daniel Zubieta, Vivek K. Kashyap, Anupam Dhasmana, Bilal B. Hafeez, Sheema Khan, Fathi T. Halaweish, Subhash C. Chauhan, Meena Jaggi. Attenuation of β-Catenin signaling by ormeloxifene in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 952.

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

Pancreatic cancer is the worst exocrine gastrointestinal cancer leading to the highest mortality. Recent studies reported that aberrant expression of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is involved in uncontrolled cell growth. However, the molecular mechanism of APE1 biological role remains unrevealed in pancreatic cancer progression. Here, we demonstrate that APE1 accelerates pancreatic cancer cell proliferation through glial cell line-derived neurotrophic factor (GDNF)/glial factor receptor &alpha, 1 (GFR&alpha, 1)/Src/ERK axis-cascade signaling. The proliferation of endogenous APE1 expressed-MIA PaCa-2, a human pancreatic carcinoma cell line, was increased by treatment with GDNF, a ligand of GFR&alpha, 1. Either of downregulated APE1 or GFR&alpha, 1 expression using small interference RNA (siRNA) inhibited GDNF-induced cancer cell proliferation. The MEK-1 inhibitor PD98059 decreased GDNF-induced MIA PaCa-2 cell proliferation. Src inactivation by either its siRNA or Src inhibitor decreased ERK-phosphorylation in response to GDNF in MIA PaCa-2 cells. Overexpression of GFR&alpha, 1 in APE1-deficient MIA PaCa-2 cells activated the phosphorylation of Src and ERK. The expression of both APE1 and GFR&alpha, 1 was gradually increased as progressing pancreatic cancer grades. Our results highlight a critical role for APE1 in GDNF-induced pancreatic cancer cell proliferation through APE1/GFR&alpha, 1/Src/ERK axis-cascade signaling and provide evidence for future potential therapeutic drug targets for the treatment of pancreatic cancer.

Pancreatic cancer (PanCa) is one of the leading causes of death from cancer in the United States. The current standard treatment for pancreatic cancer is gemcitabine, but its success is poor due to the emergence of drug resistance. Natural products have been widely investigated as potential candidates in cancer therapies, and cucurbitacin D (Cuc D) has shown excellent anticancer properties in various models. However, there is no report on the therapeutic effect of Cuc D in PanCa. In the present study, we investigated the effects of the Cuc D on PanCa cells in vitro and in vivo. Cuc D inhibited the viability of PanCa cells in a dose and time dependent manner, as evident by MTS assays. Furthermore, Cuc D treatment suppressed the colony formation, arrest cell cycle, and decreased the invasion and migration of PanCa cells. Notably, our findings suggest that mucin 13 (MUC13) is down-regulated upon Cuc D treatment, as demonstrated by Western blot and qPCR analyses. Furthermore, we report that the treatment with Cuc D restores miR-145 expression in PanCa cells/tissues. Cuc D treatment suppresses the proliferation of gemcitabine resistant PanCa cells and inhibits RRM1/2 expression. Treatment with Cuc D effectively inhibited the growth of xenograft tumors. Taken together, Cuc D could be utilized as a novel therapeutic agents for the treatment/sensitization of PanCa.

The goal of this investigation was to develop and demonstrate a polymer/paclitaxel self-assembly (PTX-SA) formulation. Polymer/PTX-SAs were screened based on smaller size of formulation using dynamic light scattering analysis. Additionally, fluorescence microscopy and flow cytometry studies exhibited that polyvinylpyrrolidone (PVP)-based PTX-SAs (PVP/PTX-SAs) had superior cellular internalization capability in MCF7 and MDA-MB-231 breast cancer cells. The optimized PVP/PTX-SAs exhibited less toxicity to human red blood cells indicating a suitable formulation for reducing systemic toxicity. The formation of PVP and PTX self-assemblies was confirmed using fluorescence quenching and transmission electron microscopy which indicated that the PVP/PTX-SAs were spherical in shape with an average size range of 53.81 nm as detected by transmission electron microscopy (TEM). FTIR spectral analysis demonstrates incorporation of polymer and paclitaxel functional groups in PVP/PTX-SAs. Both proliferation (MTS) and clonogenic (colony formation) assays were used to validate superior anticancer activity of PVP/PTX-SAs in breast cancer cells over paclitaxel. Such superior anticancer activity was also demonstrated by downregulation of the expression of pro-survival protein (Bcl-xL), upregulation of apoptosis-associated proteins (Bid, Bax, cleaved caspase 7, and cleaved PARP) and β-tubulin stabilization. These results support the hypothesis that PVP/PTX-SAs improved paclitaxel delivery to cancer cells., Graphical abstract An optimized polyvinylpyrrolidone/paclitaxel self-assembly formulation was established and tested for its improved cellular binding and anti-cancer activity against breast cancer cells.fx1

This study examines the experiences of female teachers working in private schools in Islamabad, Pakistan. These schools were divided into two categories: elite schools and non- elite schools. This differentiation was made based on fee structure, location of schools and salaries offered to teachers. Thirty-six in-depth interviews were conducted, including 24 interviews from teachers and 12 from school principals. Additionally, the responses of 96 teachers were taken through a survey in the selected schools. The data reveals that female teachers in both elite and non-elite schools encountered numerous constraints. The major issues highlighted by the teachers included low wages, lack of support from administration, disrespectful behaviour from students and job dissatisfaction. The majority considered teaching as a temporary arrangement while they waited for better employment opportunities in other sectors. This study recommends that to rescue the profession of teaching, the state can play a role by safeguarding the rights of teachers. The focus should not be merely on privatization of the education sector but due accountability of private schools is needed not only in terms of quality of education provided, but also workplace ethics and resources provided to teachers.