Silencing of PTK7 in Colon Cancer Cells: Caspase-10-Dependent Apoptosis via Mitochondrial Pathway

Protein tyrosine kinase-7 (PTK7) is a catalytically inactive receptor tyrosine kinase (RTK). PTK7 is upregulated in many common human cancers, including colon cancer, lung cancer, gastric cancer and acute myeloid leukemia. The reason for this up-regulation is not yet known. To explore the functional role of PTK7, the expression of PTK7 in HCT 116 cells was examined using small interference (siRNA)-mediated gene silencing. Following transfection, the siRNA successfully suppressed PTK7 mRNA and protein expression. Knocking down of PTK7 in HCT 116 cells inhibited cell proliferation compared to control groups and induced apoptosis. Furthermore, this apoptosis was characterized by decreased mitochondrial membrane potential and activation of caspase-9 and -10. Addition of a caspase-10 inhibitor totally blocked this apoptosis, suggesting that caspase-10 may play a critical role in PTK7-knockdown-induced apoptosis, downstream of mitochondria. These observations may indicate a role for PTK7 in cell proliferation and cell apoptosis and may provide a potential therapeutic pathway for the treatment of a variety of cancers

Health data ecosystem in Pakistan: a multisectoral qualitative assessment of needs and opportunities

Objective: Data are essential for tracking and monitoring of progress on health-related sustainable development goals (SDGs). But the capacity to analyse subnational and granular data is limited in low and middle-income countries. Although Pakistan lags behind on achieving several health-related SDGs, its health information capacity is nascent. Through an exploratory qualitative approach, we aimed to understand the current landscape and perceptions on data in decision-making among stakeholders of the health data ecosystem in Pakistan. Design: We used an exploratory qualitative study design. Setting: This study was conducted at the Aga Khan University, Karachi, Pakistan. Participants: We conducted semistructured, in-depth interviews with multidisciplinary and multisectoral stakeholders from academia, hospital management, government, Non-governmental organisations and other relevant private entities till thematic saturation was achieved. Interviews were recorded and transcribed, followed by thematic analysis using NVivo. Results: Thematic analysis of 15 in-depth interviews revealed three major themes: (1) institutions are collecting data but face barriers to its effective utilisation for decision-making. These include lack of collection of needs-responsive data, lack of a gender/equity in data collection efforts, inadequate digitisation, data reliability and limited analytical ability; (2) there is openness and enthusiasm for sharing data for advancing health; however, multiple barriers hinder this including appropriate regulatory frameworks, platforms for sharing data, interoperability and defined win-win scenarios; (3) there is limited capacity in the area of both human capital and infrastructure, for being able to use data to advance health, but there is appetite to improve and invest in capacity in this area. Conclusions: Our study identified key areas of focus that can contribute to orient a national health data roadmap and ecosystem in Pakista

Involvement of mitochondrial pathway in apoptosis induced by PTK7 scilencing.

<p>(A) Fluorescence microscope detection of mitochondrial membrane potential in treated HCT 116 cells. (B) Flow cytometry detection of mitochondrial membrane potential in treated HCT 116 cells. (C) Activation of caspase-9 involved in apoptosis induced by knocking down PTK7. The membrane was stripped and reprobed by β-actin antibody, as a loading control.</p

Cell viability after incubation with caspase inhibitors prior to transfaction of PTK7 siRNA.

<p>(A) Apoptosis induced by knocking down PTK7 was caspase-dependent. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (B) Caspase-10 inhibitor totally blocked the apoptosis induced by knock down of PTK7. Data: mean±s.d. of three independent experiments, *Student's t-test: P<0.05.</p

Mitochondria and caspase-10 involvement in the apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.

<p>(A) Fluorescence microscope detection of mitochondrial membrane potential in treated p53-null HCT 116 cells. (B) Flow cytometry detection of mitochondrial membrane potential in treated p53-null HCT 116 cells. (C) Cell viability after incubation with caspase inhibitors prior to transfaction of PTK7 siRNA. Caspase-10 inhibitor totally blocked the apoptosis induced by knock down of PTK7. Data: mean±s.d. of three independent experiments, *Student's t-test: P<0.05.</p

The activation of caspase-10 in apoptosis induced by knocking down of PTK7.

<p>(A) Western blot analysis of procaspase-10 and Bid in HCT 116 cells transfected by PTK7 siRNAs. The membrane was stripped and reprobed by β-actin antibody, as a loading control. (B) Caspase-10 activity in HCT 116 cells: untreated and treated with vehicle, nonspecific siRNA and siRNA. Results were given as ratios to caspase-10 activity in untreated cells. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05.</p

PTK7 expression in HCT 116 cells after treatment with vehicle, nonspecific siRNA and PTK7 siRNA.

<p>(A) Flow cytometry assay for the binding of the PE-labeled anti-PTK7 with HCT 116 cells (Grey curves). The black curves represent the background binding of anti-IgG-PE. The concentration of the antibody in the binding buffer was 2 µg/µL. (B) Western blot analysis of PTK7 in HCT 116 cells transfected by PTK7 siRNAs. The membrane was stripped and reprobed by β-actin antibody as a loading control. (C) Suppression of PTK7 mRNA expression in HCT 116 cells by PTK7 siRNAs. Cells were harvested after 48 h of treatment. RT-PCR was performed using gene-specific primers. The amount of PTK7 mRNA expression was normalized to the untreated group. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05.</p

PTK7 expression and cell apoptosis induced by knocking down of PTK7 in p53-null HCT 116 cells.

<p>(A) Flow cytometry assay for the binding of the PE-labeled anti-PTK7 with p53-null HCT 116 cells (Grey curves). The black curves represent the background binding of anti-IgG-PE. The concentration of the antibody in the binding buffer was 2 µg/µL. (B) The number of live p53-null HCT 116 cells was counted on day 4 after treatment with vehicle, nonspecific siRNA and PTK7 siRNA. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (C) BrdU incorporation relative to untreated cells detected by flow cytometry. p53-null HCT 116 Cells were incubated with 10 µM BrdU for 2 h after 48 h of treatment. The amount of BrdU incorporation was normalized to the untreated group. Data are mean±s.d. of three independent experiments. *Student's t-test: P<0.05. (D) Apoptosis occurrence in p53-null HCT 116 cells detected by Annexin V/PI stain on days 4 after transfection. Cells stained negative for both Annexin V and PI were considered healthy and percentage was shown in the figure.</p

National cervical cancer burden estimation through systematic review and analysis of publicly available data in Pakistan

Abstract Background Cervical cancer is a major cause of cancer-related deaths among women worldwide. Paucity of data on cervical cancer burden in countries like Pakistan hamper requisite resource allocation. Objective To estimate the burden of cervical cancer in Pakistan using available data sources. Methods We performed a systematic review to identify relevant data on Pakistan between 1995 to 2022. Study data identified through the systematic review that provided enough information to allow age specific incidence rates and age standardized incidence rates (ASIR) calculations for cervical cancer were merged. Population at risk estimates were derived and adjusted for important variables in the care-seeking pathway. The calculated ASIRs were applied to 2020 population estimates to estimate the number of cervical cancer cases in Pakistan. Results A total of 13 studies reported ASIRs for cervical cancer for Pakistan. Among the studies selected, the Karachi Cancer Registry reported the highest disease burden estimates for all reported time periods: 1995–1997 ASIR = 6.81, 1998–2002 ASIR = 7.47, and 2017–2019 ASIR = 6.02 per 100,000 women. Using data from Karachi, Punjab and Pakistan Atomic Energy Cancer Registries from 2015–2019, we derived an unadjusted ASIR for cervical cancer of 4.16 per 100,000 women (95% UI 3.28, 5.28). Varying model assumptions produced adjusted ASIRs ranging from 5.2 to 8.4 per 100,000 women. We derived an adjusted ASIR of 7.60, (95% UI 5.98, 10.01) and estimated 6166 (95% UI 4833, 8305) new cases of cervical cancer per year. Conclusion The estimated cervical cancer burden in Pakistan is higher than the WHO target. Estimates are sensitive to health seeking behavior, and appropriate physician diagnostic intervention, factors that are relevant to the case of cervical cancer, a stigmatized disease in a low-lower middle income country setting. These estimates make the case for approaching cervical cancer elimination through a multi-pronged strategy