Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer.

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

A Pilot Study of Paricalcitol plus Nanoliposomal Irinotecan and 5-FU/LV in Advanced Pancreatic Cancer Patients after Progression on Gemcitabine-Based Therapy.

PURPOSE: Vitamin D analogues remodel the desmoplastic stroma, and improve vascularity and efficacy of chemotherapy in preclinical pancreas cancer models. PATIENTS AND METHODS: We conducted a pilot study to evaluate the safety and preliminary efficacy of the vitamin D analogue paricalcitol in combination with nanoliposomal irinotecan (Nal-iri) plus 5-fluorouracil/leucovorin (5-FU/LV) in patients with advanced pancreatic cancer who had progressed on gemcitabine-based therapy. Two dose levels (DL) of paricalcitol were tested: fixed dose weekly (75 mcg, DL1) and weight-based weekly (7 mcg/kg, /DL2). The primary endpoint was safety, and secondary endpoints included overall response rate, progression-free survival (PFS), and overall survival (OS). Correlative objectives aimed to identify molecular predictors of response and alterations in the tumor stroma. RESULTS: Twenty patients (10 each in DL1 and DL2) enrolled between March 2019 and May 2021. No grade 3/4 adverse events related to paricalcitol were observed. The most common toxicities were nausea, diarrhea and fatigue, which were similar in both cohorts. Three patients discontinued study after one cycle and were not radiographically evaluable. Of the remaining 17 evaluable patients, 2 had partial response and 12 had stable disease. The median PFS for response-evaluable patients in DL1 was 4.14 months, for DL2 was 4.83 months. Intent-to-treat median OS was 6.15 and 6.66 months for DL1 and DL2, respectively. Correlative studies showed increased tumor vascularity in posttreatment samples in patients receiving the higher dose of paricalcitol (DL2). CONCLUSIONS: Paricalcitol at 7 mcg/kg/week in combination with Nal-iri/ 5-FU/LV is safely tolerated, may increase tumor vascularity and warrants further investigation.

Exploration of cytotoxic potential and tubulin polymerization inhibition activity of cis-stilbene-1,2,3-triazole congeners.

To scrutinize cis-stilbene based molecules with potential anticancer and tubulin polymerization inhibition activity, a new series of cis-stilbene-1,2,3-triazole congeners was designed and synthesized via a click chemistry protocol. The cytotoxicity of these compounds 9a-j and 10a-j was screened against lung, breast, skin and colorectal cancer cell lines. Based on the results of MTT assay, we further evaluated the selectivity index of the most active compound 9j (IC50 3.25 ± 1.04 µM on HCT-116) by comparing its IC50 value (72.24 ± 1.20 µM) to that of the normal human cell line. Further, to confirm apoptotic cell death, cell morphology and staining studies (AO/EB, DAPI and Annexin V/PI) were carried out. The outcomes of studies showed apoptotic features like change in cell shape, cornering of nuclei, micronuclei formation, fragmented, bright, horseshoe-shaped nuclei, etc. Moreover, active compound 9j displayed G2/M phase cell cycle arrest with significant tubulin polymerization inhibition activity with an IC50 value of 4.51 µM. Additionally, in silico ADMET, molecular docking and molecular dynamic studies of 9j with 3E22 protein proved the binding of the compound at the colchicine binding site of tubulin.

Acetaminophen induced hepatotoxicity: An overview of the promising protective effects of natural products and herbal formulations.

BACKGROUND: The liver plays an important role in regulating the metabolic processes and is the most frequently targeted organ by toxic chemicals. Acetaminophen (APAP) is a well-known anti-allergic, anti-pyretic, non-steroidal anti-inflammatory drug (NSAID), which upon overdose leads to hepatotoxicity, the major adverse event of this over-the-counter drug. PURPOSE: APAP overdose induced acute liver injury is the second most common cause that often requires liver transplantation worldwide, for which N-acetyl cysteine is the only synthetic drug clinically approved as an antidote. So, it was felt that there is a need for the novel therapeutic approach for the treatment of liver diseases with less adverse effects. This review provides detailed analysis of the different plant extracts; phytochemicals and herbal formulations for the amelioration of APAP-induced liver injury. METHOD: The data was collected using different online resources including PubMed, ScienceDirect, Google Scholar, Springer, and Web of Science using keywords given below. RESULTS: Over the past decades various reports have revealed that plant-based approaches may be a better treatment choice for the APAP-induced hepatotoxicity in pre-clinical experimental conditions. Moreover, herbal compounds provide several advantages over the synthetic drugs with fewer side effects, easy availability and less cost for the treatment of life-threatening diseases. CONCLUSION: The current review summarizes the hepatoprotective effects and therapeutic mechanisms of various plant extracts, active phytoconstituents and herbal formulations with potential application against APAP induced hepatotoxicity as the numbers of hepatoprotective natural products are more without clinical relativity. Further, pre-clinical pharmacological research will contribute to the designing of natural products as medicines with encouraging prospects for clinical application.

COVID-19 and fibrosis: Mechanisms, clinical relevance, and future perspectives.

Coronavirus 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has had significant impacts worldwide since its emergence in December, 2019. Despite a high recovery rate, there is a growing concern over its residual, long-term effects. However, because of a lack of long-term data, we are still far from establishing a consensus on post-COVID-19 complications. The deposition of excessive extracellular matrix (ECM), known as fibrosis, has been observed in numerous survivors of COVID-19. Given the exceptionally high number of individuals affected, there is an urgent need to address the emergence of fibrosis post-COVID-19. In this review, we discuss the clinical relevance of COVID-19-associated fibrosis, the current status of antifibrotic agents, novel antifibrotic targets, and challenges to its management.

Oncogenic KRAS-Induced Feedback Inflammatory Signaling in Pancreatic Cancer: An Overview and New Therapeutic Opportunities.

Pancreatic ductal adenocarcinoma (PDAC) remains highly refractory to treatment. While the KRAS oncogene is present in almost all PDAC cases and accounts for many of the malignant feats of PDAC, targeting KRAS or its canonical, direct effector cascades remains unsuccessful in patients. The recalcitrant nature of PDAC is also heavily influenced by its highly fibro-inflammatory tumor microenvironment (TME), which comprises an acellular extracellular matrix and various types of non-neoplastic cells including fibroblasts, immune cells, and adipocytes, underscoring the critical need to delineate the bidirectional signaling interplay between PDAC cells and the TME in order to develop novel therapeutic strategies. The impact of tumor-cell KRAS signaling on various cell types in the TME has been well covered by several reviews. In this article, we critically reviewed evidence, including work from our group, on how the feedback inflammatory signals from the TME impact and synergize with oncogenic KRAS signaling in PDAC cells, ultimately augmenting their malignant behavior. We discussed past and ongoing clinical trials that target key inflammatory pathways in PDAC and highlight lessons to be learned from outcomes. Lastly, we provided our perspective on the future of developing therapeutic strategies for PDAC through understanding the breadth and complexity of KRAS and the inflammatory signaling network.

Design, synthesis, and biological evaluation of N-(4-substituted)-3-phenylisoxazolo[5,4-d]pyrimidin-4-amine derivatives as apoptosis-inducing cytotoxic agents.

A library of new 3-phenylisoxazolo[5,4-d]pyrimidines (8-10) was designed based on a scaffold hybridization technique incorporating the important pharmacophoric features of 4-aminopyrimidine and phenyl isoxazole scaffold which is renowned for its BET inhibition activity. The designed molecules were synthesized and evaluated with the NCI-60 cell line panel. Examination by NCI-60 cell lines at single-dose and the five-dose study showed that compound 10h exhibited promising growth inhibitory effects with GI50 values on various cancer cell lines such as HCT-15 (Colon Cancer)-0.0221 µM, MDA-MB-435 (Melanoma) - 0.0318 µM, SNB-75(CNS Cancer)-0.0263 µM, and MCF7 (Breast Cancer)-0.0372 µM. Further studies to know the mechanism of action of 10h based on the phase-contrast microscopic evaluation, DAPI, acridine orange/ethidium bromide (AO/EB) staining, and annexin V-FITC assays revealed that elevation in the intracellular ROS leads to alteration in mitochondrial membrane potential which in turn induced the apoptosis in BT-474 cancer cells, which could be the plausible mechanism of action for compound 10h.

Inhibition of discoidin domain receptors by imatinib prevented pancreatic fibrosis demonstrated in experimental chronic pancreatitis model.

Discoidin domain receptors (DDR1 and DDR2) are the collagen receptors of the family tyrosine kinases, which play significant role in the diseases like inflammation, fibrosis and cancer. Chronic pancreatitis (CP) is a fibro-inflammatory disease in which recurrent pancreatic inflammation leads to pancreatic fibrosis. In the present study, we have investigated the role of DDR1 and DDR2 in CP. The induced expression of DDR1 and DDR2 was observed in primary pancreatic stellate cells (PSCs) and cerulein-induced CP. Subsequently, the protective effects of DDR1/DDR2 inhibitor, imatinib (IMT) were investigated. Pharmacological intervention with IMT effectively downregulated DDR1 and DDR2 expression. Further, IMT treatment reduced pancreatic injury, inflammation, extracellular matrix deposition and PSCs activation along with inhibition of TGF-ß1/Smad signaling pathway. Taken together, these results suggest that inhibition of DDR1 and DDR2 controls pancreatic inflammation and fibrosis, which could represent an attractive and promising therapeutic strategy for the treatment of CP.

Molecular updates on berberine in liver diseases: Bench to bedside.

Liver diseases are life-threatening illnesses and are the major cause of mortality and morbidity worldwide. These may include liver fibrosis, liver cirrhosis, and drug-induced liver toxicity. Liver diseases have a wide prevalence globally and the fifth most common cause of death among all gastrointestinal disorders. Several novel therapeutic approaches have emerged for the therapy of liver diseases that may provide better clinical outcomes with improved safety. The use of phytochemicals for the amelioration of liver diseases has gained considerable popularity. Berberine (BBR), an isoquinoline alkaloid of the protoberberine type, has emerged as a promising molecule for the treatment of gastrointestinal disorders. Accumulating studies have proved the hepatoprotective effects of BBR. BBR has been shown to modulate multiple signaling pathways implicated in the pathogenesis of liver diseases including Akt/FoxO2, PPAR-γ, Nrf2, insulin, AMPK, mTOR, and epigenetic pathways. In the present review, we have emphasized the important pharmacological activities and mechanisms of BBR in liver diseases. Further, we have reviewed various pharmacokinetic and toxicological barriers of this promising phytoconstituent. Finally, formulation-based novel approaches are also summarized to overcome the clinical hurdles for BBR.

Borneol protects against cerulein-induced oxidative stress and inflammation in acute pancreatitis mice model.

Borneol is a commonly used flavouring substance in traditional Chinese medicine, which possesses several pharmacological activities including analgesic, antiinflammatory, and antioxidant properties. The aim of this study was to investigate the effects of borneol on cerulein-induced acute pancreatitis (AP) model. Swiss albino mice were pretreated with borneol (100 and 300 mg/kg) daily for 7 days, before six consecutive injections of cerulein (50 µg/kg/hr, intraperitoneally). The protective effect of borneol was studied by biochemical, enzyme linked immunosorbent assay, histological, immunoblotting, and immunohistochemical analysis. Oral administration of borneol significantly attenuated pancreatic damage by reducing amylase, lipase levels and histological changes. Borneol attenuated cerulein-induced oxidative-nitrosative stress by decreasing malondialdehyde, nitrite levels, and elevating reduced glutathione levels. Pancreatic inflammation was ameliorated by inhibiting myeloperoxidase activity and pro-inflammatory cytokine (Interleukins and TNF-α) levels. Furthermore, borneol administration significantly increased nuclear factor E2-related factor 2 (Nrf2), superoxide dismutase (SOD1) expression and reduced phospho-NF-κB p65 expression. Treatment with borneol significantly inhibited TNF-α, IL-1ß, IL-6, and inducible nitric oxide synthase expression in cerulein-induced AP mouse model. Together, these results indicate that borneol which is currently used as US-FDA approved food adjuvant has the potential to attenuate cerulein-induced AP possibly by reducing the oxidative damage and pancreatic inflammation by modulating Nrf2/NF-κB pathway.

Nimbolide ameliorates pancreatic inflammation and apoptosis by modulating NF-κB/SIRT1 and apoptosis signaling in acute pancreatitis model.

Acute pancreatitis (AP) is a potential gastrointestinal problem most commonly associated with pancreatic inflammation and acinar cells injury. Nimbolide (NB), isolated from the tree Azadirachta indica, possesses antioxidant and anti-inflammatory effects. Here, we aimed to investigate the pancreatic protective effects of NB in ameliorating cerulein-induced pancreatic inflammation and apoptosis in AP model and evaluate the potential mechanism of action. AP was induced in Swiss albino mice by six-hourly intraperitoneal exposures of cerulein (50 µg/kg/hr) and pre-treatment of NB (0.3 and 1 mg/kg) 7 days prior to the cerulein exposure. Various parameters associated with AP in plasma and pancreatic tissues were evaluated. Severity of AP was effectively ameliorated by NB as shown by reducing pancreatic edema, plasma amylase and lipase levels, MPO levels and in cerulein-induced histological damage. Further, the antioxidant effect of NB was associated with a significant inhibition of oxidative-nitrosative stress in Raw 264.7 cells and cerulein-induced AP mice. Moreover, NB suppressed proinflammatory cytokines, iNOS and nitrotyrosine expression. In addition, NB inhibited NF-κB activation and increased SIRT1 expression in cerulein challenged mice. Furthermore, NB also inhibited pancreatic apoptosis by downregulating cleaved caspase 3 and Bax while upregulating Bcl2 expression in cerulein-treated mice. Inhibition of pancreatic inflammation and apoptosis resulted in attenuation of cerulein-induced AP. These results suggest that NB exerts strong anti-pancreatitis effects against cerulein-induced AP by combating inflammatory and apoptosis signaling via SIRT1 activation.

Piperlongumine attenuates bile duct ligation-induced liver fibrosis in mice via inhibition of TGF-ß1/Smad and EMT pathways.

Liver fibrosis (LF) is a progressive liver injury that may result in excessive accumulation of extracellular matrix (ECM). However, transforming growth factor-beta (TGF-ß) and epithelial to mesenchymal transition (EMT) play a central role in the progression of LF through the activation of matrix producing hepatic stellate cells (HSCs). Piperlongumine (PL), an alkaloid extracted from Piper longum, has been reported to possess anti-inflammatory and antioxidant activities in various diseases but its hepatoprotective and antifibrotic effects have not been reported yet. Therefore, in the present study, we investigated the protective effect of PL in bile duct ligation (BDL)-induced LF model and explored the molecular mechanisms underlying its antifibrotic effect. BDL group displayed a significant degree of liver damage, oxidative-nitrosative stress, hepatic inflammation and collagen deposition in the liver while these pathological changes were effectively attenuated by treatment with PL. Furthermore, we found that PL treatment greatly inhibited HSCs activation and ECM deposition via downregulation of fibronectin, α-SMA, collagen1a, and collagen3a expression in the fibrotic livers. We further demonstrated that PL administration significantly inhibited TGF-ß1/Smad and EMT signaling pathways. Our study demonstrated that PL exerted strong hepatoprotective and antifibrotic activities against BDL-induced LF and might be an effective therapeutic agent for the treatment of LF.

Berberine attenuates severity of chronic pancreatitis and fibrosis via AMPK-mediated inhibition of TGF-ß1/Smad signaling and M2 polarization.

Berberine (BR) acts as an AMP-activated protein kinase (AMPK) activator which possesses antioxidant and anti-inflammatory properties. In this study, we have investigated the effects of BR against cerulein-induced chronic pancreatitis (CP) via inhibition of TGF-ß/Smad signaling and M2 macrophages polarization in AMPK dependent manner. Cerulein-induced CP mice were treated with BR (3 and 10 mg/kg), intraperitoneally every day for 21 days. Our results indicated that, BR treatment (10 mg/kg) significantly reduced oxidative-nitrosative stress, histological alterations, inflammatory cells infiltration and collagen deposition in pancreatic tissue. BR treatment also prevented cerulein-induced pancreatic stellate cells (PSCs) activation and extracellular matrix (ECM) deposition via downregulation of α-SMA, collagen1a, collagen3a and fibronectin expression. Mechanistically, treatment with BR significantly activated AMPK signaling as compared to cerulein-challenged mice. Further, administration of BR also inhibited TGF-ß/Smad signaling and macrophages polarization in cerulein-induced CP in-vivo models and TGF-ß1 stimulated RAW 264.7 macrophages in-vitro. Together, our results strongly suggest that BR treatment protected against cerulein-induced CP and associated fibrosis progression by inhibiting TGF-ß1/Smad signaling and M2 macrophages polarization in an AMPK dependent manner.

Nimbolide abrogates cerulein-induced chronic pancreatitis by modulating ß-catenin/Smad in a sirtuin-dependent way.

Chronic pancreatitis (CP) is one of the leading causes of mortality worldwide with no clinically approved therapeutic interventions. The present study was designed to investigate the protective effect of nimbolide (NB), an active constituent of neem tree (Azadirachta indica), by targeting ß-catenin/Smad/SIRT1 in cerulein-induced CP model. The effects of NB was investigated on cerulein (50 µg/kg/hr*6 exposures /day, 3 days a week for 3 weeks) induced CP in mice. Amylase and lipase activity were measured and histopathological evaluation was performed. Collagen deposition in the pancreatic tissue was estimated by hydroxyproline assay, and collagen specific staining picrosirius red and Masson's trichrome. Cerulein-induced CP was significantly controlled by NB treatment, as shown by the downregulation of ß-catenin/Smad signaling in a SIRT1 dependent manner. NB treatment significantly decreased α-SMA, MMP-2, collagen1a, fibronectin, TGF-ß1, p-Smad-2/3 expression and extracellular matrix (ECM) deposition in pancreatic tissue. However, the protective effects of NB on cerulein-induced CP were undermined by nicotinamide (NMD) or splitomicin, sirtuin 1 (SIRT1) inhibitors treatment. NB treatment modulated protein expression by activating SIRT1 and decreasing the expression of ß-catenin/Smad proteins in CP mice. However, the expression of SIRT1 in pancreatic tissue was elevated by NB treatment and it was decreased by NMD or splitomicin treatment. In summary, our results strongly suggest that NB exerted promising protective effects in cerulein-induced CP model by inhibiting ß-catenin/Smad in a sirtuin-dependent manner, which could be attributed to its anti-inflammatory and antifibrotic effects. Our study suggests that NB could be an effective therapeutic intervention for the treatment of CP.

Crocin, an active constituent of Crocus sativus ameliorates cerulein induced pancreatic inflammation and oxidative stress.

Acute pancreatitis (AP) is a disorder of the pancreas marked by profound inflammation and oxidative stress. Phytoconstituents presents an important toolbox of preventive strategies to combat inflammatory disorders. To this end, we selected the active constituent of Crocus sativus, crocin for evaluation against cerulein-induced AP, owing to its promising antiinflammatory activity in acute as well as chronic inflammatory conditions. The animals were randomly divided into five groups comprising of normal control, cerulein control, crocin low dose (30 mg/kg), crocin high dose (100 mg/kg), and crocin control (100 mg/kg). Various biochemical parameters and the levels of inflammatory cytokines and p65-NFκB were measured. The mechanism was investigated by histology and immunohistochemistry. We found that crocin significantly reduced the pancreatic edema, amylase, and lipase levels. It abrogated the oxidative stress incurred by cerulein challenge. We found that crocin modulated the pancreatic inflammatory cytokine levels. Crocin perturbed the nuclear translocation of p65-NFκB. Crocin reverted the pancreatic histology associated with AP. Furthermore, it upregulated the expression of Nrf-2 and downregulated the expression of IL-6, TNF-α, nitrotyrosine, and NFκB. Cumulatively, these results indicate that crocin has promising potential to prevent cerulein induced AP and regular intake of saffron can prove beneficial for the pancreatic health.

Cerulein-induced chronic pancreatitis in Swiss albino mice: An improved short-term model for pharmacological screening.

There is a need for short-term, reliable and reproducible animal model of chronic pancreatitis (CP) in small animals like mice. This study was aimed to establish the 9 exposures of cerulein-induced CP in mice. Repeated intraperitoneal cerulein injections were performed at 6 consecutive doses (50 µg/kg)/day, 3 days a week for 3 weeks to induce chronic pancreatitis in Swiss albino mice. The severity of damage was assessed by biochemical assays and histopathology. The expression of pro-inflammatory cytokine and fibrotic proteins was assessed by IHC and western blotting. The cerulein treated mice showed significantly elevated plasma amylase (p < .0285) and lipase levels (p < .0022) and resulted in significantly increased pancreatic oxidative (p < .0022) and nitrosative (p < .0022) stress. The hydroxyproline levels were 3.06 fold increased in the cerulein treated mice. The expressions of fibrotic cytokine TGF-ß1 by 1.8 folds and pro-inflammatory cytokines TNF-α by 2.3 fold, IL-6 by 2.2 fold and IL-1ß by 3.7 fold were markedly increased in cerulein treated mice. The histological evaluations indicated increased inflammatory cells infiltration and deposition of collagen. Moreover, the expression of fibrotic markers such as α-SMA increased by 2.5 folds (p < .00014), collagen1a by 1.3 folds (p < .0258) and fibronectin by 3.5 folds (p < .00014) were significantly increased. Our study demonstrates the superiority of 9 exposures of cerulein-induced CP model in mice with the reduction of duration, cerulein exposure, more economical and mortality rate of mice over the available models. Therefore, our model may be suitable to evaluate the pharmacological effects of new drugs in chronic pancreatitis.