Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

The Differential Clinical Impacts of Cachexia and Sarcopenia on the Prognosis of Advanced Pancreatic Cancer.

Pancreatic cancer (PC) has the highest frequency of developing cancer cachexia (CC)-sarcopenia (SC) syndrome, which negatively influences patients' outcome, quality of life, and tolerance/response to treatments. However, the clinical impacts of CC, SC, and their associated factors on outcomes for advanced PC has yet to be fully investigated. A total of 232 patients were enrolled in this study for the retrospective review of their clinical information and the measurement of skeletal muscle areas at the third lumber vertebra by computed tomography scan to identify CC or SC. The association and concurrent occurrence of clinicopathological features in each patient, prevalence rates, and prognosis with the CC or SC were calculated. CC and SC were observed in 83.6% (n = 194) and 49.1% (n = 114) of PC patients, respectively. Low hemoglobin levels more often occurred in CC patients than in non-CC patients (p = 0.014). Older age (p = 0.000), female gender (p = 0.024), low body mass index (BMI) values (p = 0.004), low hemoglobin levels (p = 0.036), and low albumin levels (p = 0.001) were more often found in SC patients than in non-SC patients. Univariate and multivariate analyses showed that CC was an independent poor prognostic factor of overall survival (OS) and progression-free survival for all patients, the chemotherapy (C/T) subgroup, and the high BMI subgroup. Meanwhile, SC was an independent predictor of poor OS for the subgroups of C/T or high BMI but not for all patients. These findings reveal the clinical differences for CC and SC and provide useful information for predicting the prognosis of advanced PC patients and conducting personalized medicine.

Anti-cancer therapeutic benefit of red guava extracts as a potential therapy in combination with doxorubicin or targeted therapy for triple-negative breast cancer cells.

Guava extracts purified from leaf and bark have many bio-active molecules with anti-cancer activities. In addition, lycopene-rich extracts obtained from red guava fruit can induce apoptosis in estrogen receptor-positive breast cancers. Triple-negative breast cancer (TNBC) lacks estrogen receptors, progesterone receptors and human epidermal growth factor receptor 2 (HER2) and, therefore, hormone therapy and targeted therapy are not used in the clinic. The purpose of this study was to determine whether red guava fruit extracts can affect the proliferation of TNBC cells. In this study, cell viability was determined by using the MTT assay. Apoptosis and necrosis were analyzed using flow cytometry. Cleaved caspase-3 and PARP were analyzed by western blotting. We found that red guava extracts can, through caspase-3 activation and PARP cleavage signaling, induce apoptotic and necrotic death in TNBC cells. Our results thus show the therapeutic benefit of red guava extracts as a potential cancer treatment for TNBC in combination with doxorubicin or targeted therapy.

Anti-Cancer Effects of Sulfasalazine and Vitamin E Succinate in MDA-MB 231 Triple-Negative Breast Cancer Cells.

Aim: Sulfasalazine (SSZ) displayed anti-cancer activities. Vitamin E succinate (VES) could inhibit cell growth in various cancer cells. However, chemical therapies were often not useful for triple-negative breast cancer cells (TNBCs) treatment. Here, this study investigated the anti-cancer effects and the mechanisms on TNBCs under combination treatment with SSZ and VES. Methods: Cell viability was analyzed by using the MTT assay. The H2O2 levels were determined by using lucigenin-amplified chemiluminescence method. In addition, caspase and MAPs signals were studied by using western blotting. Results: Low-dose VES antagonized the SSZ-induced cytotoxicity effects while high-dose VES promoted the SSZ-induced cytotoxicity effects on TNBCs. In addition, SSZ alone treatment activated both caspase-3 and ERK signals, however, VES alone treatment only activated JNK signals. On the other hand, activation of caspase-3, JNK, and ERK were found in SSZ plus VES-treated cells. Conclusion: Combined SSZ and VES has synergistic or antagonistic cytotoxic effects depending on VES concentration. In addition, different cytotoxic signals are induced on SSZ-treated, VES-treated and SSZ plus VES-treated cells.

Extracts from guava fruit protect renal tubular endothelial cells against acetaminophen­induced cytotoxicity.

Acetaminophen (APAP) is an analgesic and antipyretic agent primarily used in the clinical setting. However, high doses of APAP can cause oxidative stress. Guavas have been reported to provide anti­inflammatory, anti­microbial, anti­oxidative and anti­diarrheal functions. In addition, guavas have been reported to prevent renal damage due to progression of diabetes mellitus. Therefore, the aim of the present study was to investigate whether guavas can reduce APAP­induced renal cell damage. In the present study, extracts from guavas were obtained and added to APAP­treated renal tubular endothelial cells. The present results demonstrated that APAP induces cytotoxicity in renal tubular endothelial cells, while guava extracts inhibited this cytotoxicity. In addition, the study demonstrated that the protective effects of guava extracts against APAP­induced cytotoxicity may be associated with inhibition of oxidative stress and caspase­3 activation.

Combined treatment with vitamin C and methotrexate inhibits triple-negative breast cancer cell growth by increasing H2O2 accumulation and activating caspase-3 and p38 pathways.

Methotrexate (MTX) is widely used as both an anticancer and anti-rheumatoid arthritis drug. Although MTX has been used to inhibit the growth of many cancer cells, it cannot effectively inhibit growth of triple-negative breast cancer cells (TNBC cells). Vitamin C is an antioxidant that can prevent oxidative stress. In addition, vitamin C has been applied as adjunct treatment for growth inhibition of cancer cells. Recent studies indicated that combined treatment with vitamin C and MTX may inhibit MCF-7 and MDA-MB-231 breast cancer cell growth through G2/M elongation. However, the mechanisms remain unknown. The aim of the present study was to determine whether combined treatment with low-dose vitamin C and MTX inhibits TNBC cell growth and to investigate the mechanisms of vitamin C/MTX-induced cytotoxicity. Neither low-dose vitamin C alone nor MTX alone inhibited TNBC cell growth. However, combined low-dose vitamin C and MTX had synergistic anti-proliferative/cytotoxic effects on TNBC cells. In addition, co-treatment increased H2O2 levels and activated both caspase-3 and p38 cell death pathways.

Extracts containing CLPs of Bacillus amyloliquefaciens JN68 isolated from chicken intestines exert antimicrobial effects, particularly on methicillin-resistant Staphylococcus aureus and Listeria monocytogenes.

Bacillus amyloliquefaciens JN68, which has been discussed with regards to its antimicrobial activities, was successfully isolated from healthy chicken intestines in the present study. Using the spot-on-the-lawn antagonism method, the preliminary study indicated that a suspension culture of the B. amyloliquefaciens JN68 strain can inhibit the growth of Aspergillus niger and Penicillium pinophilum. Furthermore, the cyclic lipopeptides (CLPs) produced by the B. amyloliquefaciens JN68 strain were further purified through acid precipitation and Bond Elut®C18 chromatography, and their structures were identified using the liquid chromatography­electrospray ionization­mass spectrometry (MS)/MS method. Purified CLPs exerted broad spectrum antimicrobial activities on various pathogenic and foodborne bacteria and fungi, as determined using the agar well diffusion method. Listeria monocytogenes can induce listeriosis, which is associated with a high mortality rate. Methicillin­resistant Staphylococcus aureus (MRSA) is a major pathogenic bacteria that causes nosocomial infections. Therefore, L. monocytogenes and MRSA are currently of great concern. The present study aimed to determine whether B. amyloliquefaciens JN68 extracts could inhibit L. monocytogenes and MRSA. The results indicated that extracts of B. amyloliquefaciens JN68 have CLP components, and can successfully inhibit the growth of L. monocytogenes and MRSA.

Combined treatment with vitamin E and gefitinib has synergistic effects to inhibit TGF-ß1-induced renal fibroblast proliferation.

Renal fibroblast proliferation is key in renal fibrosis and chronic kidney disease. Transforming growth factor-ß1 (TGF-ß1) has been demonstrated to be an important factor that induces cell proliferation in renal fibroblasts. Epidermal growth factor receptor (EGFR) is also recognized as a factor promoting renal fibroblast proliferation. In addition, mitogen­activated protein kinase signaling pathways are associated with TGF­ß1­ and EGFR­induced cell proliferation. Gefitinib, an EGFR tyrosine kinase inhibitor, is predominantly used as an anti­tumor therapeutic agent in clinical therapeutic strategies. However, gefitinib has been suggested to exert anti­proliferative effects on renal fibroblasts, however, high­dose gefitinib may result in serious side effects. The present study aims to determine whether low­dose gefitinib reduces gefitinib­induced side effects and maintains the anti­proliferative effects on renal fibroblasts. TGF­ß1 promotes cell proliferation in renal fibroblasts, and the current study demonstrates that low­dose gefitinib treatment exhibits anti­proliferative effects similar to those of high­dose gefitinib treatment. Thus, although high­dose gefitinib is a conventional anti­tumor drug, low­dose gefitinib may be of use in renal fibrosis treatment. Furthermore, the present study demonstrates that a combined treatment with low-dose gefitinib and vitamin E has synergistic effects that reduce TGF­ß1­induced fibroblast proliferation, cell-cycle arrest and the ERK phosphorylation pathway.

Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation.

Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high­dose APAP­induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low­dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high­dose APAP treatment inhibited while therapeutic and low­dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase­9/­3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low­dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts.

RC-6 ribonuclease induces caspase activation, cellular senescence and neuron-like morphology in NT2 embryonal carcinoma cells.

Frog ribonucleases have been demonstrated to have anticancer activities. However, whether RC-6 ribonuclease exerts anticancer activity on human embryonal carcinoma cells remains unclear. In the present study, RC-6 induced cytotoxicity in NT2 cells (a human embryonal carcinoma cell line) and our studies showed that RC-6 can exert anticancer effects and induce caspase-9 and -3 activities. Moreover, to date, there is no evidence that frog ribonuclease-induced cytotoxicity effects are related to cellular senescence. Therefore, our studies showed that RC-6 can increase p16 and p21 protein levels and induce cellular senescence in NT2 cells. Notably, similar to retinoic acid-differentiated NT2 cells, neuron-like morphology was found on some remaining live cells after RC-6 treatment. In conclusion, our study is the first to demonstrate that RC-6 can induce cytotoxic effects, caspase-9/-3 activities, cellular senescence and neuron-like morphology in NT2 cells.