Long-term di-(2-ethylhexyl) phthalate exposure reduces sorafenib treatment efficacy by enhancing mesenchymal transition in hepatocellular carcinoma.

Di(2-ethylhexyl) phthalate (DEHP) is a worldwide common plasticizer. Nevertheless, DEHP is easily leached out to the environment due to the lack of covalent bonds with plastic. High dose of DEHP exposure is often observed in hemodialysis patients because of the continual usage of plastic medical devices. Although the liver is the major organ that catabolizes DEHP, the impact of long-term DEHP exposure on the sensitivity of liver cancer to chemotherapy remains unclear. In this study, we established long-term DEHP-exposed hepatocellular carcinoma (HCC) cells and two NOD/SCID mice models to investigate the effects and the underlying mechanisms of long-term DEHP exposure on chemosensitivity of HCC. The results showed long-term DEHP exposure potentially increased epithelial-mesenchymal transition (EMT) in HCC cells. Next generation sequencing showed that long-term DEHP exposure increased cell adhesion/migratory related genes expression and blunted sorafenib treatment induced genes alterations. Long-term exposure to DEHP reduced the sensitivity of HCC cells to sorafenib-induced anti-migratory effect by enhancing the EMT transcription factors (slug, twist, and ZEB1) and mesenchymal protein (vimentin) expression. In NOD/SCID mice model, we showed that long-term DEHP-exposed HCC cells exhibited higher growth rate. Regarding the anti-HCC effects of sorafenib, subcutaneous HuH7 tumor grew slowly in sorafenib-treated mice. Nonetheless, the anti-tumor growth effect of sorafenib was not observed in long-term DEHP-exposed mice. Higher mesenchymal markers and proliferating cell nuclear antigen (PCNA) expression were found in sorafenib-treated long-term DEHP-exposed mice. In conclusion, long-term DEHP exposure promoted migratory activity in HCC cells and decreased sorafenib sensitivity in tumor-bearing mice.

Scutellarin, a flavonoid compound from Scutellaria barbata, suppresses growth of breast cancer stem cells in vitro and in tumor-bearing mice.

BACKGROUND: Scutellaria barbata D. Don (SB), commonly known as Ban Zhi Lian and firstly documented by Shigong Chen, is a dried whole plant that has been studied for its therapeutic effects on breast cancer, colon cancer, and prostate cancer. Among its various compounds, scutellarin (SCU) has been demonstrated with anti-tumor effects. PURPOSE: This study aimed to evaluate the effects of SB water extract (SBW) and scutellarin on breast cancer stem cells (BCSCs), and to investigate their potential therapeutic effects on breast tumors in mice. METHODS: BCSCs were enriched from human breast cancer cells (MDA-MB-231 and MDA-MB-361) and their characteristics were analyzed. The effects of varying concentrations of SBW and scutellarin on cell viability, proliferation, self-renewal, and migration abilities were studied, along with the underlying mechanisms. The in vivo anti-tumor effects of scutellarin were further evaluated in SCID/NOD mice. Firstly, mice were inoculated with naïve BCSCs and subjected to treatment with scutellarin or vehicle. Secondly, BCSCs were pre-treated with scutellarin or vehicle prior to inoculation into mice. RESULTS: The derived BCSCs expressed CD44, CD133 and ALDH1, but not CD24, indicating that BCSCs have been successfully induced from both MDA-MB-231 and MDA-MB-361 cells. Both SBW and scutellarin reduced the viability, proliferation, sphere and colony formation, and migration of BCSCs. In mice with tumors derived from naïve BCSCs, scutellarin significantly reduced tumor growth, expression of proliferative (Ki67) and stem cell markers (CD44), and lung metastasis. In addition, pre-treatment with scutellarin also slowed tumor growth. Western blot results suggested the involvement of Wnt/ß-catenin, NF-κB, and PTEN/Akt/mTOR signaling pathways underlying the inhibitory effects of scutellarin. CONCLUSION: Our study demonstrated for the first time that both SB water extract and scutellarin could reduce the proliferation and migration of BCSCs in vitro. Scutellarin was shown to possess novel inhibitory activities in BCSCs progression. These findings suggest that Scutellaria barbata water extract, in particular, scutellarin, may have potential to be further developed as an adjuvant therapy for reducing breast cancer recurrence.

Anti-Colorectal Cancer Effects of Fucoidan Complex-Based Functional Beverage Through Retarding Proliferation, Cell Cycle and Epithelial-Mesenchymal Transition Signaling Pathways.

BACKGROUND: Fucus vesiculosus-derived fucoidan, a multifunctional bioactive polysaccharide sourced from marine organisms, exhibits a wide range of therapeutic properties, including its anti-tumor effects. While previous research has reported on its anti-cancer potential, limited studies have explored its synergistic capabilities when combined with other natural bioactive ingredients. In this current study, we present the development of an integrative functional beverage, denoted as VMW-FC, which is composed of a fucoidan complex (FC) along with a blend of various herbal components, including vegetables (V), mulberries and fruits (M), and spelt wheat (W). OBJECTIVE: Colorectal cancer (CRC) remains a significant cause of mortality, particularly in metastatic cases. Therefore, the urgent need for novel alternative medicines that comprehensively inhibit CRC persists. In this investigation, we assess the impact of VMW-FC on CRC cell proliferation, cell cycle dynamics, metastasis, in vivo tumorigenesis, and potential side effects. METHODS: Cell growth was assessed using MTT and colony formation assays, while metastatic potential was evaluated through wound healing and transwell migration assays. The underlying signaling mechanisms were elucidated through qPCR and western blot analysis. In vivo tumor formation and potential side effects were evaluated using a subcutaneous tumor-bearing NOD/SCID mouse model. RESULTS: Our findings demonstrate that VMW-FC significantly impedes CRC proliferation and migration in a dose- and time-dependent manner. Furthermore, it induces sub-G1 cell cycle arrest and an increase in apoptotic cell populations, as confirmed through flow-cytometric analysis. Notably, VMW-FC also suppresses xenograft tumor growth in NOD/SCID mice without causing renal or hepatic toxicity. CONCLUSION: The integrative herbal concoction VMW-FC presents a promising approach for inhibiting CRC by slowing proliferation and migration, inducing cell cycle arrest and apoptosis, and suppressing markers associated with proliferation (Ki-67, PCNA, and CDKs) and epithelial-mesenchymal transition (EMT) (Vimentin, N-cadherin, and ß-catenin).

Bright ferritin for long-term MR imaging of human embryonic stem cells.

BACKGROUND: A non-invasive imaging technology that can monitor cell viability, retention, distribution, and interaction with host tissue after transplantation is needed for optimizing and translating stem cell-based therapies. Current cell imaging approaches are limited in sensitivity or specificity, or both, for in vivo cell tracking. The objective of this study was to apply a novel ferritin-based magnetic resonance imaging (MRI) platform to longitudinal tracking of human embryonic stem cells (hESCs) in vivo. METHODS: Human embryonic stem cells (hESCs) were genetically modified to stably overexpress ferritin using the CRISPR-Cas9 system. Cellular toxicity associated with ferritin overexpression and manganese (Mn) supplementation were assessed based on cell viability, proliferation, and metabolic activity. Ferritin-overexpressing hESCs were characterized based on stem cell pluripotency and cardiac-lineage differentiation capability. Cells were supplemented with Mn and imaged in vitro as cell pellets on a preclinical 3 T MR scanner. T1-weighted images and T1 relaxation times were analyzed to assess contrast. For in vivo study, three million cells were injected into the leg muscle of non-obese diabetic severe combined immunodeficiency (NOD SCID) mice. Mn was administrated subcutaneously. T1-weighted sequences and T1 mapping were used to image the animals for longitudinal in vivo cell tracking. Cell survival, proliferation, and teratoma formation were non-invasively monitored by MRI. Histological analysis was used to validate MRI results. RESULTS: Ferritin-overexpressing hESCs labeled with 0.1 mM MnCl2 provided significant T1-induced bright contrast on in vitro MRI, with no adverse effect on cell viability, proliferation, pluripotency, and differentiation into cardiomyocytes. Transplanted hESCs displayed significant bright contrast on MRI 24 h after Mn administration, with contrast persisting for 5 days. Bright contrast was recalled at 4-6 weeks with early teratoma outgrowth. CONCLUSIONS: The bright-ferritin platform provides the first demonstration of longitudinal cell tracking with signal recall, opening a window on the massive cell death that hESCs undergo in the weeks following transplantation before the surviving cell fraction proliferates to form teratomas.

Significant expansion of the donor pool achieved by utilizing islets of variable quality in the production of allogeneic "Neo-Islets", 3-D organoids of Mesenchymal Stromal and islet cells, a novel immune-isolating biotherapy for Type I Diabetes.

Novel biotherapies for Type 1 Diabetes that provide a significantly expanded donor pool and that deliver all islet hormones without requiring anti-rejection drugs are urgently needed. Scoring systems have improved islet allotransplantation outcomes, but their use may potentially result in the waste of valuable cells for novel therapies. To address these issues, we created "Neo-Islets" (NIs), islet-sized organoids, by co-culturing in ultralow adhesion flasks culture-expanded islet (ICs) and Mesenchymal Stromal Cells (MSCs) (x 24 hrs, 1:1 ratio). The MSCs exert powerful immune- and cyto-protective, anti-inflammatory, proangiogenic, and other beneficial actions in NIs. The robust in vitro expansion of all islet hormone-producing cells is coupled to their expected progressive de-differentiation mediated by serum-induced cell cycle entry and Epithelial-Mesenchymal Transition (EMT). Re-differentiation in vivo of the ICs and resumption of their physiological functions occurs by reversal of EMT and serum withdrawal-induced exit from the cell cycle. Accordingly, we reported that allogeneic, i.p.-administered NIs engraft in the omentum, increase Treg numbers and reestablish permanent normoglycemia in autoimmune diabetic NOD mice without immunosuppression. Our FDA-guided pilot study (INAD 012-0776) in insulin-dependent pet dogs showed similar responses, and both human- and canine-NIs established normoglycemia in STZ-diabetic NOD/SCID mice even though the utilized islets would be scored as unsuitable for transplantation. The present study further demonstrates that islet gene expression profiles (α, ß, γ, δ) in human "non-clinical grade" islets obtained from diverse, non-diabetic human and canine donors (n = 6 each) closely correlate with population doublings, and the in vivo re-differentiation of endocrine islet cells clearly corresponds with the reestablishment of euglycemia in diabetic mice. Conclusion: human-NIs created from diverse, "non-clinical grade" donors have the potential to greatly expand patient access to this curative therapy of T1DM, facilitated by the efficient in vitro expansion of ICs that can produce ~ 270 therapeutic NI doses per donor for 70 kg recipients.

MET Inhibits the Proliferation of EC Cells by Increasing MPA Sensitivity.

Context: The high resistance rate and high recurrence rate of progesterone only as a treatment for endometrial cancer (EC) limit its clinical application. Metformin (MET) may have antitumor ability. Combining MET and medroxyprogesterone acetate (MPA) may strengthen their inhibitory effects on proliferation of EC cells, but MET's mechanisms remain unclear. Objective: The study intended to identify the specific molecular mechanism that MET combined with MPA uses against EC progression. Design: The research team performed a controlled animal study. Setting: The study took place at Xuzhou Medical University in Xuzhou, China. Animals: The animals were16 female non-obese diabetic-severe combined immunodeficient (NOD-SCID) nude mice, about 12 to 16 g in weight. Interventions: The research team divided randomly, the mice into four groups and induced EC in all groups, four in each group: (1) The control group which received received normal saline, (2) the MPA group, which received 100 mg/kg of MPA; (3) the MET group, which received metformin at the rate of 200 mg/kg, each gavage volume was 0.1ml; (4) the MET+MPA group, which received 100 mg/kg of MPA and 200 mg/kg of MET. Outcome measures: The research team: (1) used a CCK-8 kit, an EdU assay, and a flow-cytometry assay to measure cancer-cell proliferation, count, and viability; determine the cell cycle; and measure apoptosis; (2) performed a Western blot analysis to determine the expression of the PR, CD133, pAkt, totalAkt, p-mTOR, and totalTOR antibodies; and (3) determined the size and volume of tumors in vivo and used immunohistochemical staining to determine expression of the Ki67 protein. Results: The MET+MPA group had a significantly lower number of cancer cells than the MET or MDA groups (both P < .001). That group also had significantly more stagnated cancer cells in the G0/G1 phase and significantly fewer cancer cells in the S phase or G2/M phase control, MET, or MPA groups (all P < .01). The MET+MPA group's PCNA and Ki-67 protein expression was significantly lower than that of the MET and MPA group. The EDU assay yielded similar results. Additionally, the MET+MPA group had significantly higher PR expression than that of to MET or MPA group (both P < .001). The MET and MPA groups' expression of CD133, p-Akt, and p-mTOR were significantly lower than those of the control group, while the MET+MPA group's levels were significantly lower than those of the MET and MPA groups. In-vivo experiments revealed that the MET and MPA groups did show decreased tumor size and volume. The MET+MPA group had tumor weights that were significantly lower and tumor volumes were significantly smaller than those of the MET and MPA groups (all P < .001). Immunohistochemical analysis revealed that the MET+MPA group's levels of the Ki-67 antigen were significantly lower than those of the MET and MPA groups. Conclusions: MET inhibited the proliferation of EC cells by increasing MPA-sensitivity, which was dependent on the inhibition of the CD133 expression and the Akt/mTOR pathway. In addition, if MET acts as an effective progestin sensitizer, it certainly offers promising therapeutic prospects for patients with early-stage EC or overgrown endometrium who have fertility requirements.

Ginsenoside 24-OH-PD from red ginseng inhibits acute T-lymphocytic leukaemia by activating the mitochondrial pathway.

Ginsenoside 24-hydroxy-ginsengdiol (24-OH-PD), extracted from red ginseng, is a novel diol-type ginsenoside, strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia (T-ALL) CCRF-CEM cells. Our research aimed at investigating the mechanism underlying this inhibition. Cell viability was determined using the cell counting kit-8 (CCK-8) assay, and NOD/SCID mice bearing CCRF-CEM cells were used to verify the therapeutic effect of 24-OH-PD on T-ALL in vivo. We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis. Cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) levels were detected by flow cytometry. The activity of caspase3 and caspase9 was detected by enzyme activity detection kits. The expression levels of apoptosis-related proteins and mRNA were determined through western blotting and quantitative reverse-transcription PCR assays (qRT-PCR). CCK-8 assay and animal xenograft experiments confirmed that 24-OH-PD significantly inhibited T-ALL in a dose-dependent manner, both in vivo and in vitro. RNA-Seq results suggest that mitochondria-mediated apoptosis pathway plays an important role in this process. Furthermore, intracellular ROS levels increased, mPTP opened, and ΔΨm decreased following 24-OH-PD treatment. Pretreatment with the antioxidant, NAC, reversed the effects of 24-OH-PD on apoptosis and ROS generation. Moreover, 24-OH-PD treatment increased the expression of Bax and caspase family members, thereby releasing cytochrome c (Cytc) and inducing apoptosis. Our findings showed that, 24-OH-PD induces apoptosis in CCRF-CEM cells by activating the mitochondrial-dependent apoptosis pathway through ROS accumulation. This inhibitory effect implies that 24-OH-PD could be further developed as treatment of T-ALL.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3ß/STAT3 signaling axis.

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3ßSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3ß, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3ß/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Toxic profile of marinobufagin from poisonous Amazon toads and antitumoral effects on human colorectal carcinomas.

ETHNOPHARMACOLOGICAL RELEVANCE: South Americans natives have extensively used the toad "kururu" to reduce/treat skin infections, cutaneous lesions and sores. They release secretions rich in bufadienolides, polyhydroxy steroids with well-documented cardiotonic and antiproliferative actions, but in vivo antitumoral evaluations in mammals are rare, and toxicological safety has been left in second place. AIMS OF THE STUDY: This investigation used in silico, in vitro and in vivo tools to evaluate acute and subacute toxic effects of marinobufagin and the anticancer action in tumor-bearing mice models. MATERIALS AND METHODS: Initially, in silico toxic predictions were performed, followed by in vitro assays using human and murine normal and tumor lines. Next, acute and subacute studies on mice investigated the behavior, hematological and intestinal transit profile and antitumoral activity of marinobufagin in sarcoma 180- and HCT-116 colorectal carcinoma-transplanted mice for 7 and 15 days, respectively. Ex vivo and in vivo cytogenetic assays in Sarcoma 180 and bone marrow cells and histopathological examinations were also executed. RESULTS: In silico studies revealed ecotoxicological effects on crustaceans (Daphnia sp.), fishes (Pimephales promelas and Oryzias latipes), and algae. A 24-h marinobufagin-induced acute toxicity included signals of central activity, mainly (vocal frenzy, absence of body tonus, increased ventilation, ataxia, and equilibrium loss), and convulsions and death at 10 mg/kg. The bufadienolide presented effective in vitro cytotoxic action on human lines of colorectal carcinomas in a similar way to ouabain and tumor reduction in marinobufagin-treated SCID-bearing HCT-116 heterotopic xenografts. Animals under subacute nonlethal doses exhibited a decrease in creatinine clearance with normal levels of blood urea, probably as a result of a marinobufagin-induced renal perfusion fall. Nevertheless, only minor morphological side effects were identified in kidneys, livers, hearts and lungs. CONCLUSIONS: Marinobufagin has in vitro and in vivo anticancer action on colorectal carcinoma and mild and reversible alterations in key metabolic organs without direct chemotherapy-induced gastrointestinal effects at subacute exposure, but it causes acute ataxia, equilibrium loss, convulsions and death at higher acute exposure.

LncFALEC recruits ART5/PARP1 and promotes castration-resistant prostate cancer through enhancing PARP1-meditated self PARylation.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are abnormal expression in various malignant tumors. Our previous research demonstrated that focally amplified long non-coding RNA (lncRNA) on chromosome 1 (FALEC) is an oncogenic lncRNA in prostate cancer (PCa). However, the role of FALEC in castration-resistant prostate cancer (CRPC) is poorly understood. In this study, we showed FALEC was upregulated in post-castration tissues and CRPC cells, and increased FALEC expression was associated with poor survival in post-castration PCa patients. RNA FISH demonstrated FALEC was translocated into nucleus in CRPC cells. RNA pulldown and followed Mass Spectrometry (MS) assay demonstrated FALEC directly interacted with PARP1 and loss of function assay showed FALEC depletion sensitized CRPC cells to castration treatment and restored NAD+. Specific PARP1 inhibitor AG14361 and NAD+ endogenous competitor NADP+ sensitized FALEC-deleted CRPC cells to castration treatment. FALEC increasing PARP1 meditated self PARylation through recruiting ART5 and down regulation of ART5 decreased CRPC cell viability and restored NAD+ through inhibiting PARP1meditated self PARylation in vitro. Furthermore, ART5 was indispensable for FALEC directly interaction and regulation of PARP1, loss of ART5 impaired FALEC and PARP1 associated self PARylation. In vivo, FALEC depleted combined with PARP1 inhibitor decreased CRPC cell derived tumor growth and metastasis in a model of castration treatment NOD/SCID mice. Together, these results established that FALEC may be a novel diagnostic marker for PCa progression and provides a potential new therapeutic strategy to target the FALEC/ART5/PARP1 complex in CRPC patients.

Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor.

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

AGA induces sub-G1 cell cycle arrest and apoptosis in human colon cancer cells through p53-independent/p53-dependent pathway.

BACKGROUND: Despite the advancement in chemotherapeutic drugs for colon cancer treatment, it is still a life-threatening disease worldwide due to drug resistance. Therefore, an urgently needed to develop novel drugs for colon cancer therapies. AGA is a combination of traditional Chinese medicine Antler's extract (A), Ganoderma lucidum (G), and Antrodia camphorata (A); it contains a lot of biomolecules like polysaccharides, fatty acids, and triterpenoids that are known to exerting anti-oxidative, anti-inflammatory, anti-microbial and anti-tumor activities in oral cancer. In this study, we investigate AGA anti-proliferative, anti-metastatic and apoptotic activity to explore its anti-cancer activity against colon cancer cells and its underlying mechanism. METHOD: Here, in-vitro studies were performed to determine the antiproliferative activity of AGA through MTT and colony formation assays. Wound healing and transwell migration assay were used to evaluate the metastasis. Flow cytometry and protein expression were used to investigate the involved molecular mechanism by evaluating the cell cycle and apoptosis. The in-vivo anti-cancerous activity of AGA was assessed by xenograft mice model of colon cancer cells. RESULTS: We found that AGA significantly inhibited the proliferative capacity and metastasis of colon cancer cells in-vitro. In addition, AGA induced cell cycle arrest in the sub-G1 phase through upregulating p21 and downregulating CDK2, CDK6 in SW620, and CDK4 in SW480 and HT29, respectively. Annexin-v assay indicated that colon cancer cells had entered early and late apoptosis after treatment with AGA. Furthermore, a mechanistic protein expressions study revealed that AGA in p53-dependent and independent regulated the apoptosis of colon cancer by downregulating the p53 protein expression in SW620 and SW480 cells but upregulating in a dose-dependent manner in HT29 cells and increasing the expression of Bax and caspase-9 to inhibit the colon cancer cells. In vivo study, we found that AGA significantly reduced the xenograft tumor growth in NOD/SCID mice with no adverse effect on the kidney and liver. CONCLUSION: Collectively, AGA has the potential to inhibit colon cancer through inhibiting proliferation, migration, and cell cycle kinase by upregulating p21 protein expression and promoting the apoptotic protein in a p53-dependent and independent manner.

Human growth hormone supplement promotes human lymphohematopoietic cell reconstitution in immunodeficient mice.

Aim: To investigate the potential of human growth hormone (hGH) to improve human hematopoietic reconstitution in humanized mice. Materials & methods: Immunodeficient mice were conditioned by total body irradiation and transplanted with human CD34+ fetal liver cells. Peripheral blood, spleen and bone marrow were harvested, and levels of human lymphohematopoietic cells were determined by flow cytometry. Results: Supplementation with hGH elevated human lymphohematopoietic chimerism by more than twofold. Treatment with hGH resulted in significantly increased reconstitution of human B cells and myeloid cells in lymphoid organs, enhanced human erythropoiesis in the bone morrow, and improved engraftment of human hematopoietic stem cells. Conclusion: hGH supplementation promotes human lymphohematopoietic reconstitution in humanized mice.

Humanized mice generated by human hematopoietic stem cell transplantation play crucial roles in biomedical investigations. One of the factors hindering the efficacy of their construction is the lack of or insufficient interaction of human cells to mouse cytokines and growth hormones (GHs) that are crucial for hematopoiesis and immune cell differentiation. In this study, we show that injection of human GH significantly improved human hematopoietic stem cell engraftment and function, as well as immune cell reconstitution in humanized mice. Our findings indicate that human cells may not efficiently respond to mouse GH, and generation of immunodeficient mice producing human GH may improve the efficacy of humanized mouse construction.

San-Zhong-Kui-Jian-Tang Exerts Antitumor Effects Associated With Decreased Cell Proliferation and Metastasis by Targeting ERK and the Epithelial-Mesenchymal Transition Pathway in Oral Cavity Squamous Cell Carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is an aggressive cancer whose 5-year survival rate remains poor. San-Zhong-Kui-Jian-Tang (SZKJT), a Chinese herbal formula, has long been used in clinical practice as adjuvant therapy in cancers. However, its therapeutic effects and molecular mechanisms in OSCC remain unclear. METHODS: We investigated the potential therapeutic effects and molecular mechanism of SZKJT in OSCC in tumor cell lines and in tumor xenograft mice and evaluated combined SZKJT and cisplatin treatment efficacy. In vitro-cultured OSCC cells were administered SZKJT at different doses or SZKJT plus cisplatin, and cell proliferation, colony formation assays, and cell cycle analysis were used to assess the effects on cancer cell proliferation and apoptosis. We also analyzed the effects of SZKJT on oral cancer cell line migration, the regulation of mitogen-activated protein kinase (MAPK) signaling, and epithelial-mesenchymal transition (EMT)-associated genes. The antitumor effects of SZKJT plus cisplatin were also tested in vivo using a tumor-bearing NOD/SCID mice model. RESULTS: The results showed that SZKJT effectively inhibited OSCC cell proliferation, induced cell cycle S phase arrest, and induced cell apoptosis. SZKJT also inhibited cell migration by modulating the MAPK signaling and epithelial-mesenchymal transition (EMT) pathway. Further exploration suggested that SZKJT affects OSCC by modulating ERK pathway; downregulating vimentin, fibronectin, and Oct-4; and upregulating E-cadherin. In vivo, SZKJT significantly inhibited tumor growth, and SZKJT and cisplatin exerted synergistic antitumor effects in model animals. CONCLUSIONS: SZKJT exerts antitumor effects in OSCC cells. Additionally, SZKJT and cisplatin exhibit synergy in OSCC treatment. These findings support the clinical usage of Chinese herbal formulas as adjuvant therapy with chemotherapy in cancer treatment.

Chloroxylon swietenia (Roxb.) DC induces cell death and apoptosis by down-regulating the NF-κB pathway in MCF-7 breast cancer cells: In vitro and in vivo investigations.

BACKGROUND: Natural products with targeted bioactivity have gained major attention in the field of cancer research owing to emerging anti-cancer drug resistance and off target toxicities. Chloroxylon swietenia (Roxb.) DC is recognized as a folklore medicinal plant and has numerous therapeutic benefits in the folklore medicine system, however the anti-cancer potential of this plant and its mechanism of action is poorly understood. AIMS: The aim of the study was to investigate the anti-breast cancer efficacy of C. swietenia leaves methanol extract (CSLME) against MCF-7 hormone dependent human breast cancer cell line with possible mechanism of action. METHODS AND RESULTS: The anti-breast cancer activity of CSLME against MCF-7 cells was assessed by evaluating its efficacy toward cytotoxicity, cell migration, colony formation, DNA fragmentation, apoptosis, cytoskeleton, angiogenesis, cell cycle regulation, and animal toxicity. The preliminary screening of CSLME against MCF-7 cells revealed the cytotoxicity (IC50 20 µg/ml), inhibited cell migration, colony formation, and angiogenesis. It was observed that CSLME induces apoptosis by nuclear fragmentation and disruption of cytoskeleton by actin derangement. The results of Annexin V-FITC assay and cell cycle analysis by flow cytometry clearly pointed out the sizable fraction of apoptotic cells, and arrested the cells at G2/M phase of cell cycle. The results of the immunoblotting experiments showed that CSLME activates intrinsic pathway of apoptosis with down regulation of anti-apoptotic marker like Bcl2, up regulation of pro-apoptotic markers like Bax & Bad, along with successful cleavage of Caspase-9 and PARP-1. Further, western blot analysis revealed the possible down regulation of NF-κB pathway by CSLME, which may be responsible for anti-cancer activity in MCF-7 cells. In vivo animal model studies using NOD-SCID mice demonstrated impressive anti-tumor activity with significant reduction in tumor volume of MCF-7 tumor xenograft. Of note, in-vivo acute oral toxicity study as per Organization for Economic Cooperation and Development 423 revealed the nontoxic nature of CSLME. CONCLUSION: The in vitro and in vivo findings clearly outline the potential of CSLME as inhibitor of growth and proliferation of MCF-7 cells. Mechanistically, CSLME seems to activate intrinsic pathway of apoptosis, arrest cell cycle, target actin cytoskeleton, inhibit growth, colony formation, migration, and angiogenesis, with down regulation of NF-κB pathway leading to cell death.

Redox-responsive nanoparticles enhance radiation therapy by altering multifaceted radio-resistance mechanisms in human castration-resistant prostate cancer cells and xenografts.

INTRODUCTION: Radiation therapy (RT) is a major modality for the treatment of prostate cancer (PCa), especially castration-resistant PCa (CRPC). However, hypoxia, often seen in PCa tumors, leads to radiation-resistance. This work investigates the effect of a novel oxygen-generating polymer-lipid manganese dioxide nanoparticle (PLMDs) on improving RT outcomes in CRPC xenograft models by modulating the tumor microenvironment (TME) both before and after RT. MATERIALS AND METHODS: Human PC3 and DU145 PCa cells were used to investigate clonogenic inhibition and DNA repair pathways in vitro. Tumor hypoxia and post-RT angiogenesis were evaluated in a PC3-bearing SCID mouse model. PC3 and DU145 xenografts were used to study the efficacy of PLMD in combination with single or fractionated RT. RESULTS: PLMD plus RT significantly inhibited clonogenic potential, increased DNA double-strand breaks, and reduced DNA damage repair in hypoxic PC3 and DU145 cells as compared to RT alone. PLMD significantly reduced hypoxia-positive areas, hypoxia induced factor 1α (HIF-1α) expression, and protein carbonyl levels (a measure of oxidative stress). Application of PLMD with RT decreased RT-induced angiogenic biomarkers by up to 3-fold. Treatment of the human CRPC xenografts with PLMD plus RT (single or fractionated doses) significantly prolonged median survival of the host compared to RT alone resulting in up to a 40% curative rate. CONCLUSION: PLMD treatment modulated TME and sensitized hypoxic human CRPC cells to RT thus enhancing the efficacy of RT. These results confirmed the potential of PLMD as an adjuvant to RT for the treatment of hypoxic CRPC.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

BCL-XL inhibition induces an FGFR4-mediated rescue response in colorectal cancer.

The heterogeneous therapy response observed in colorectal cancer is in part due to cancer stem cells (CSCs) that resist chemotherapeutic insults. The anti-apoptotic protein BCL-XL plays a critical role in protecting CSCs from cell death, where its inhibition with high doses of BH3 mimetics can induce apoptosis. Here, we screen a compound library for synergy with low-dose BCL-XL inhibitor A-1155463 to identify pathways that regulate sensitivity to BCL-XL inhibition and reveal that fibroblast growth factor receptor (FGFR)4 inhibition effectively sensitizes to A-1155463 both in vitro and in vivo. Mechanistically, we identify a rescue response that is activated upon BCL-XL inhibition and leads to rapid FGF2 secretion and subsequent FGFR4-mediated post-translational stabilization of MCL-1. FGFR4 inhibition prevents MCL-1 upregulation and thereby sensitizes CSCs to BCL-XL inhibition. Altogether, our findings suggest a cell transferable induction of a FGF2/FGFR4 rescue response in CRC that is induced upon BCL-XL inhibition and leads to MCL-1 upregulation.

Targeting serine hydroxymethyltransferases 1 and 2 for T-cell acute lymphoblastic leukemia therapy.

Despite progress in the treatment of acute lymphoblastic leukemia (ALL), T-cell ALL (T-ALL) has limited treatment options, particularly in the setting of relapsed/refractory disease. Using an unbiased genome-scale CRISPR-Cas9 screen we sought to identify pathway dependencies for T-ALL which could be harnessed for therapy development. Disruption of the one-carbon folate, purine and pyrimidine pathways scored as the top metabolic pathways required for T-ALL proliferation. We used a recently developed inhibitor of SHMT1 and SHMT2, RZ-2994, to characterize the effect of inhibiting these enzymes of the one-carbon folate pathway in T-ALL and found that T-ALL cell lines were differentially sensitive to RZ-2994, with the drug inducing a S/G2 cell cycle arrest. The effects of SHMT1/2 inhibition were rescued by formate supplementation. Loss of both SHMT1 and SHMT2 was necessary for impaired growth and cell cycle arrest, with suppression of both SHMT1 and SHMT2 inhibiting leukemia progression in vivo. RZ-2994 also decreased leukemia burden in vivo and remained effective in the setting of methotrexate resistance in vitro. This study highlights the significance of the one-carbon folate pathway in T-ALL and supports further development of SHMT inhibitors for treatment of T-ALL and other cancers.

Novel colchicine derivative CR42-24 demonstrates potent anti-tumor activity in urothelial carcinoma.

Bladder cancers, and specifically urothelial carcinoma, have few effective treatment options, and tumors typically develop resistance against standard of care chemotherapies leading to significant mortality. The development of alternative therapies with increased selectivity and improved tolerability would significantly impact this patient population. Here, we investigate a novel colchicine derivative, CR42-24, with increased selectivity for the ßIII tubulin subtype as a treatment for urothelial carcinoma. ßIII tubulin is a promising target due to its low expression in healthy tissues and its clinical association with poor prognosis. This study demonstrated that CR42-24 is selectively cytotoxic to several cancer cell lines at low nanomolar IC50, with high activity in bladder cancer cell lines both in vitro and in vivo. CR42-24 monotherapy in an aggressive urothelial carcinoma xenograft model results in effective control when treated early. We observed significant ablation of large tumors and patient-derived xenografts at low doses with excellent tolerability. CR42-24 was highly synergistic in combination with the standard of care chemotherapies gemcitabine and cisplatin, further increasing its therapeutic potential as a novel treatment for urothelial carcinoma.