Paulmurugan, R. | Search bioRxiv

bioRxiv2023

Restoration of the Lost Human Beta Defensin (hBD-1) in Cancer as a Strategy to Improve the Efficacy of Chemotherapy

Both innate and adaptive immunity are the important components of the human defense system against various diseases including cancer. Human Beta Defensin (hBD-1) is one such immunomodulatory peptide which is lost at high frequencies in malignant cancers, while high levels of expression are maintained in benign regions making it a potential biomarker for the onset and metastasis of the disease. Loss of putative function of hBD-1 as a tumor suppressor gene combined with the defects in apoptosis pathways (CD95, ASK1) make tumor cells insensitive to chemotherapy and render it ineffective. Triple negative breast cancer (TNBC) is an aggressive form of breast cancer for which no targeted therapy works due to lack of biomarkers (ER, PR and HER2 negative). That makes chemotherapy as a first line of treatment despite high side effects. TNBC is known for avoiding immunosurveillance and desensitizing themselves to intervention by dysregulating cell death pathways (CD95 & ASK1) and developing resistance to chemotherapy A priori Activation of Apoptosis Pathways of Tumor often referred to as AAAPT is a novel targeted tumor sensitizing technology which sensitizes low responsive and resistant tumor cells to evoke a better response from the current treatments for TNBC. Here, we show that hBD-1 is shown to target tumor specific biomarker Trx, activates dual cell death pathways CD95 and ASK1 (apoptosis stimulating kinase) to sensitize TNBC cells to chemotherapy drug Doxorubicin. As far as we know, this is the first-time injection of hBD-1 in TNBC mouse model to prove the restoration of hBD-1 back to the basal level can sensitize cancer cells which resulted in significant reduction of tumor volume in TNBC mouse model in vivo. Sensitizing the low or non-responsive tumor cells by AAAPT and making chemotherapy work at lower doses may lead to the significant reduction of dose related side effects and may expand the therapeutic index of the current treatments.

cancer biology↗

bioRxiv2023

Enkurin: A novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been linked in recent research with the pathogenesis of myeloproliferative neoplasms (MPNs). As yet, however, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using ex vivo and in vitro systems (including CD34+ cultures from patient bone marrow, and healthy cord/peripheral blood specimens), we extend our prior data from MPN patient platelet RNA sequencing, and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelets, parent megakaryocytes, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein, implicated originally only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across MPN patient specimens and experimental models, with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR by shRNA in CD34+ derived megakaryocytes further confirm this association with CDC20 at both RNA and protein levels; and indicate a likely role for the PI3K/Akt pathway. The inverse association of ENKUR and CDC20 expression was further confirmed upon treatment with thapsigargin (an agent that causes protein misfolding in the ER by selective loss of calcium) in both megakaryocyte and platelet fractions at RNA and protein levels. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis beyond the genetic alterations; and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/523111v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@80834corg.highwire.dtl.DTLVardef@12c033forg.highwire.dtl.DTLVardef@8948c8org.highwire.dtl.DTLVardef@1096184_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIEnkurin, a calcium adaptor protein, is identified as a novel marker of pathogenesis in MPNs. C_LIO_LIMPN megakaryocyte and platelet expression of enkurin at RNA and protein levels is inversely associated with a cell differentiation cycle gene, CDC20. C_LIO_LILikely role for dysregulated calcium homeostasis, and ER and protein folding stress in MPN transformation. C_LI

cancer biology↗

bioRxiv2020

Foxa1/2 downregulates liver differentiation markers and the endoderm and liver gene regulatory network in human stem cells and in a human stable liver cell line

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA dictates human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA targeting of FOXA1 and FOXA2 in human hepatic (HepG2) cells and during hepatic differentiation significantly downregulated albumin (p < 0.05) and GRN TF gene expression (HNF4A, HEX, HNF1B, TBX3) (p < 0.05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid (GSC), FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < 0.05), all consistent with a partial/transient cell reprogramming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, and accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During hPSC-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate complex effects of FOXA1/2 on hepatic GRN effecting de-differentiation and metabolism with applications in studies of cancer, differentiation, and organogenesis.

bioengineering↗

bioRxiv2019

A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) Technology: Development of Targeted Apoptosis Initiators for Cancer Treatment .

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. The principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral damaging effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept "A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (ling cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro. At higher dose, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AMP-001 as a neoadjuvant to chemotherapy to achieve a better efficacy and reduced off-target toxicity by the current treatments. Summary StatementA Priori Activation of Apoptosis Pathways of Tumor often referred to as "AAAPT" is a novel targeted tumor sensitizing technology which synergizes with chemotherapy to enhance the treatment efficacy.

cancer biology↗

bioRxiv2019

A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) Technology: Development of Targeted Apoptosis Initiators for Cancer Treatment.

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. The principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral damaging effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept "A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (ling cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro. At higher dose, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AMP-001 as a neoadjuvant to chemotherapy to achieve a better efficacy and reduced off-target toxicity by the current treatments. Summary StatementA Priori Activation of Apoptosis Pathways of Tumor often referred to as "AAAPT" is a novel targeted tumor sensitizing technology which synergizes with chemotherapy to enhance the treatment efficacy.

cancer biology↗

bioRxiv2019

Ultrasound- and microbubble-mediated targeted delivery of therapeutic microRNA-loaded nanocarriers to deep liver and kidney tissues in pigs

In this study, we designed and validated a platform for ultrasound (US) and microbubble (MB)-mediated delivery of FDA-approved pegylated poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with anticancer microRNAs (miRNAs) to deep tissues in a pig model. Small RNAs have the ability to reprogram tumor cells and sensitize them to clinically used chemotherapy. However, to overcome their short intravascular circulation half-life and achieve controlled and sustained release into tumor cells, anticancer miRNAs need to be encapsulated into NPs. Moreover, focused US combined with gas-filled MBs provides a safe and noninvasive way to improve the permeability of tumor vasculature and increase the delivery efficiency of drug-loaded nanocarriers. A single handheld, curvilinear US array was used in this study for image-guidance and therapy with clinical-grade SonoVue MBs. First, we validated the platform on phantoms to optimize the MB cavitation dose based on acoustic parameters, including peak negative pressure, pulse length, and pulse repetition frequency. We then tested the system in vivo by delivering PLGA-NPs co-loaded with antisense-miRNA-21 and antisense-miRNA-10b in pig liver and kidney. Enhanced miRNA delivery was observed (1.9- to 3.7-fold increase) as a result of the US-MB treatment compared to untreated control regions. Additionally, we used highly fluorescent semiconducting polymer nanoparticles (SPNs) co-delivered with miRNA-loaded PLGA-NPs to visually assess NP delivery. Fluorescent microscopy of SPNs confirmed NP extravasation and showed the presence of particles in the extravascular compartment. Hematoxylin and eosin staining of treated tissues did not reveal tissue damage. The results presented in this manuscript suggest that enhanced delivery of miRNA-loaded NPs to target regions in deep organs is feasible in large animal models using the proposed platform.

bioengineering↗