Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Rapid Reduction of CEA and Stable Metastasis in an NRAS-mutant Rectal-Cancer Patient Treated With FOLFIRI and Bevacizumab Combined With Oral Recombinant Methioninase and a Low-Methionine Diet Upon Metastatic Recurrence After FOLFIRI and Bevacizumab Treatment Alone.

BACKGROUND/AIM: The choice of chemotherapy agents for RAS-mutant colorectal cancer is limited, and prognosis is poor compared to RAS-wild-type colorectal cancer. The purpose of the present study was to evaluate the effectiveness of methionine restriction combined with chemotherapy in a patient with NRAS-mutant rectal cancer. PATIENTS AND METHODS: A 59-year-old female was diagnosed with lung-metastatic recurrence of NRAS-mutant rectal cancer two and a half years after resection of the primary tumor. She started chemotherapy, which consisted of fluorouracil, irinotecan (FOLFIRI), and bevacizumab, in October 2020. Eight months later, stereotactic body radiation therapy (SBRT) was performed to treat the lung metastases. She stopped chemotherapy at this point and had blood tests and computed tomography (CT) scans regularly. Her CEA level increased to 139.91 ng/ml and her lung metastasis became larger by September 2022. Therefore, she was reintroduced to FOLFIRI and bevacizumab in October 2022, and also started a low-methionine diet and oral recombinant methioninase (o-rMETase) as a supplement. RESULTS: After starting the combination therapy with o-rMETase, a low-methionine diet, FOLFIRI, and bevacizumab, blood CEA levels very rapidly decreased and were almost within the normal limits five months later. CT findings showed the lung metastasis did not grow. CONCLUSION: Methionine restriction comprising o-rMETase and a low-methionine diet combined with first-line chemotherapy was effective in a patient with NRAS-mutant rectal cancer in which metastasis had re-occurred after first-line chemotherapy alone.

Elevated Pro-inflammatory Cytokine Levels in Acute Reflux Esophagitis Are Reduced by 1,25 Dihydroxy Vitamin D3.

BACKGROUND/AIM: Gastric acid reflux into the esophagus can cause irritation and inflammation of the esophagus and progress to reflux esophagitis (RE). Vitamin D3 (VitD3) has anti-inflammatory effects and plays an important regulatory role in adaptive and innate immunity. We hypothesized that VitD3 may play a protective role in RE. MATERIALS AND METHODS: Seventy male Sprague-Dawley rats were used, and acute RE (n=35) or chronic RE (n=35) were surgically induced. The effects of different doses of VitD3 on morphological changes and alteration of pro-inflammatory cytokine levels were examined in the rat models. Western blot analysis was performed to determine protein expression levels of IL-1ß, IL-6, and IL-8 in esophageal tissues. Serum levels of VitD3 and calcium were determined using enzyme-linked immunosorbent assays. RESULTS: The protein expression of pro-inflammatory cytokines IL-1ß, IL-6, and IL-8 was found significantly increased in RE. VitD3 treatment significantly reduced the levels of these pro-inflammatory cytokines in both the low-dose and high-dose VitD3 groups compared to control groups in acute RE, but not chronic RE. Macrographic and histopathological examination revealed various degrees of esophageal impairment in rats following surgical induction of acute or chronic RE in rats. These impairments were not improved by VitD3. Morphological grading of esophageal mucosa showed no significant differences between acute and chronic RE. Elevated serum levels of calcium were observed after VitD3 treatment. CONCLUSION: IL-1ß, IL-6, and IL-8 levels were significantly elevated in RE. The abnormal increase in these important pro-inflammatory cytokines was suppressed by VitD3 in the rat models of acute RE. These novel findings suggest a potential protective role of VitD3 in early-stage RE.

Evodiamine inhibits ESCC by inducing M-phase cell-cycle arrest via CUL4A/p53/p21 axis and activating noxa-dependent intrinsic and DR4-dependent extrinsic apoptosis.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a malignancy with high incidence in several regions of China, and the prognosis of patients with ESCC is unfavorable. Evodiamine (Evo), a small molecule derived from the traditional Chinese herb Evodia rutaecarpa, has shown anti-cancer efficacy in numerous human malignancies but not in ESCC. PURPOSE: To determine whether Evo induces cell-cycle arrest and apoptosis in ESCC in vitro and in vivo and elucidate the underlying mechanisms. METHODS: ATPlite and colony formation assay were used to validate the inhibiting effect of Evo on three ESCC cells in vitro; Two subcutaneous tumor models of ESCC cells were used to evaluate the anti-ESCC effect of Evo and assess the biosafety of Evo in vivo; RNAseq and Database of KEGG pathway analysis provided a direction for the mechanistic study of Evo; FACS was used to detect Evo-induced cell cycle arrest and cell apoptosis in ESCC cells; Western blot and QPCR were respectively used to detect the level of related genes and proteins in Evo-treated ESCC cells; SiRNA and other experimental techniques were used to identify the molecular mechanism of Evo-induced ESCC cell cycle arrest and cell apoptosis. RESULTS: Evo significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistically, Evo induced M-phase cell-cycle arrest by inactivation of CUL4A E3 ligase, which mediates degradation blockage of p53 and transcriptional activation of p21. With the prolonged treatment time, Evo triggered both Noxa-dependent intrinsic and DR4-dependent extrinsic cell apoptosis in two ESCC cell lines. CONCLUSION: Our findings revealed the anti-tumor efficacy and mechanisms of Evo, providing a solid scientific basis for Evo as an attractive choice for ESCC treatment.

Stage IV Pancreatic Cancer Patient Treated With FOLFIRINOX Combined With Oral Methioninase: A Highly-Rare Case With Long-term Stable Disease.

BACKGROUND: Pancreatic cancer is one of the most recalcitrant cancers, and more effective therapy is needed. Pre-clinical studies have shown that patient-derived orthotopic xenograft (PDOX) mouse models of pancreatic cancer are effectively treated with oral recombinant methioninase (o-rMETase). CASE REPORT: A 62-year-old woman diagnosed with stage IV pancreatic cancer was treated with the combination of 5-fluorouracil/leucovorin, irinotecan, and oxaliplatinum (FOLFIRINOX) every two weeks and o-rMETase twice a day as a supplement. The patient was also on a low-methionine diet. Disease progression was monitored by CA19-9 and computed tomography. The patient initially responded to FOLFIRINOX, shown by a great reduction in CA19-9 levels, with tumor shrinkage shown by computed tomography. The patient began taking o-rMETase and went on a low-methionine diet one year after diagnosis which she has maintained without side effects for 7 months. The patient's CA19-9 level and tumor size remain stable 19 months after diagnosis. The patient is alive and has maintained a high performance status. Historical data show that less than 5% of stage IV pancreatic-cancer patients on FOLFIRINOX have stable disease 1.5 years after diagnosis. CONCLUSION: The combination of o-rMETase and FOLFIRINOX may be synergistic in stage IV pancreatic cancer.

Andrographolide, a diterpene lactone from the Traditional Chinese Medicine Andrographis paniculate, induces senescence in human lung adenocarcinoma via p53/p21 and Skp2/p27.

BACKGROUND: Senescence leads to permanent cell-cycle arrest and is a potential target for cancer therapy. Andrographolide (AD) is a diterpene lactone isolated from Traditional Chinese Medicine (TCM) Andrographis paniculate, which has been used as an anti-inflammatory drug in clinical practice with the potential to target senescence in recalcitrant lung cancer. PURPOSE: To determine whether AD can induce senescence in human lung adenocarcinoma in vitro and in vivo and to elucidate the underlying mechanisms. METHODS: SA-ß-Gal staining was used to detect the expression of senescence-associated ß-galactosidase (SA-ß-Gal) in human lung adenocarcinoma cells A549 and NCI-H1795. DNA damage was examined by the detection of γH2AX foci. Cell cycle was analyzed by flow cytometry. Cancer cell proliferation was determined by ATPlite assay and clonogenic survival assay in vitro. Tumor growth was determined in a mouse model of A549. The expression level of proteins and mRNA was estimated by Western blotting and Quantitative RT-PCR, respectively. Small interfering RNA (siRNA) was used to knock down p21, p27 and p53 to explore the potential mechanism of AD-induced senescence in human lung adenocarcinoma cells. RESULTS: AD-induced A549 and NCI-H1795 cell senescence determined by increased cell size, flattened morphology, DNA damage, cell cycle arrest as well as the increased expression of ß-galactosidase. AD inhibited cell proliferation in lung cells in vitro and lung cells xenograft growth in nude mice. p21 and p27, the major cell cycle regulators and mediators of senescence, were upregulated at the protein level in AD-treated A549 lung adenocarcinoma in vitro and in vivo. Further studies demonstrated that AD induced cell senescence via p53/p21 and Skp2/p27. CONCLUSION: In the present study, we found that the primary anti-inflammatory drug AD could have a potential antitumor effect in lung cancer. We demonstrated that AD induced lung adenocarcinoma senescence in vitro and in vivo via p53/p21 and Skp2/p27 for the first time. AD is therefore a promising senescence-inducing therapeutic for recalcitrant human lung adenocarcinoma.

Fangchinoline Inhibits Human Esophageal Cancer by Transactivating ATF4 to Trigger Both Noxa-Dependent Intrinsic and DR5-Dependent Extrinsic Apoptosis.

Esophageal squamous cell carcinoma (ESCC) is a recalcitrant cancer. The Chinese herbal monomer fangchinoline (FCL) has been reported to have anti-tumor activity in several human cancer cell types. However, the therapeutic efficacy and underlying mechanism on ESCC remain to be elucidated. In the present study, for the first time, we demonstrated that FCL significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistic studies revealed that FCL-induced G1 phase cell-cycle arrest in ESCC which is dependent on p21 and p27. Moreover, we found that FCL coordinatively triggered Noxa-dependent intrinsic apoptosis and DR5-dependent extrinsic apoptosis by transactivating ATF4, which is a novel mechanism. Our findings elucidated the tumor-suppressive efficacy and mechanisms of FCL and demonstrated FCL is a potential anti-ESCC agent.

Immuno-hyperthermia effected by antibody-conjugated nanoparticles selectively targets and eradicates individual cancer cells.

Hyperthermia has been used for cancer therapy for a long period of time, but has shown limited clinical efficacy. Induction-heating hyperthermia using the combination of magnetic nanoparticles (MNPs) and an alternating magnetic field (AMF), termed magnetic hyperthermia (MHT), has previously shown efficacy in an orthotopic mouse model of disseminated gastric cancer. In the present study, superparamagnetic iron oxide nanoparticles (SPIONs), a type of MNP, were conjugated with an anti-HER2 antibody, trastuzumab and termed anti-HER2-antibody-linked SPION nanoparticles (anti-HER2 SPIONs). Anti-HER2 SPIONs selectively targeted HER2-expressing cancer cells co-cultured along with normal fibroblasts and HER2-negative cancer cells and caused apoptosis only in the HER2-expressing individual cancer cells. The results of the present study show proof-of-concept of a novel hyperthermia technology, immuno-MHT for selective cancer therapy, that targets individual cancer cells.Abbreviations: AMF: alternating magnetic field; DDW: double distilled water; DMEM: Dulbecco's Modified Eagle's; Medium; f: frequency; FBS: fetal bovine serum; FITC: Fluorescein isothiocyanate; GFP: green fluorescent protein; H: amplitude; Hsp: heat shock protein; MHT: magnetic hyperthermia; MNPs: magnetic nanoparticles; PI: propidium iodide; RFP: red fluorescent protein; SPION: superparamagnetic iron oxide (Fe3O4) nanoparticle.

Traditional Chinese Medicine Xihuang Wan Inhibited Lewis Lung Carcinoma in a Syngeneic Model, Equivalent to Cytotoxic Chemotherapy, by Altering Multiple Signaling Pathways.

BACKGROUND/AIM: Xihuang Wan (XHW), a traditional Chinese medicine (TCM), has been used in China for a variety of cancers including lung cancer. The present study evaluated the efficacy of XHW on a Lewis lung mouse model and explored the potential mechanism via transcriptomics. MATERIALS AND METHODS: The mice were randomized into 6 groups: 1) untreated control (n=10); 2) low-dose XHW; 3) medium-dose XHW; 4) high-dose XHW; 5) cisplatin; and 6) untreated blank (n=4). Lewis lung carcinoma (LLC) cells were injected subcutaneously except for the 4 mice in the blank group. The body weight and tumor length and width were measured every 3 days. RNA-sequencing was performed on tumors in the high-dose XHW group and the control group. RESULTS: XHW inhibited the growth of LLC in a syngeneic mouse model, without toxicity, with equivalent efficacy to cisplatin. RNA-sequencing demonstrated that many signaling pathways were involved in XHW-mediated inhibition of LLC, including tumor necrosis factor, estrogen, cyclic guanosine 3', 5'-monophosphate-protein kinase G, apelin and the peroxisome proliferator-activated receptor signaling pathways. CONCLUSION: XHW inhibited LLC carcinoma through different pathways and shows clinical promise for patients who cannot tolerate platinum-based drugs.

Hyperthermia generated by magnetic nanoparticles for effective treatment of disseminated peritoneal cancer in an orthotopic nude-mouse model.

Magnetic hyperthermia (MHT), which combines magnetic nanoparticles (MNPs) with an alternating magnetic field (AMF), holds promise as a cancer therapy. There have been many studies about hyperthermia, most of which have been performed by direct injection of MNPs into tumor tissues. However, there have been no reports of treating peritoneal disseminated disease with MHT to date. In the present study, we treated peritoneal metastasis of gastric cancer with MHT using superparamagnetic iron oxide (Fe3O4) nanoparticle (SPION) coated with carboxydextran as an MNP, in an orthotopic mouse model mimicking early peritoneal disseminated disease of gastric cancer. SPIONs of an optimal size were intraperitoneally administered, and an AMF (390 kHz, 28 kAm-1) was applied for 10 minutes, four times every three days. Three weeks after the first MHT treatment, the peritoneal metastases were significantly inhibited compared with the AMF-alone group or the untreated-control group. The results of the present study show that MHT can be applied as a new treatment option for disseminated peritoneal gastric cancer.Abbreviations: AMF: alternating magnetic field; Cy1: cytology-positive; DMEM: Dulbecco's Modified Eagle's Medium; FBS: fetal bovine serum; H&E: hematoxylin and eosin; HIPEC: hyperthermic intraperitoneal chemotherapy; MEM: Minimum Essential Medium; MHT: magnetic hyperthermia; MNPs: magnetic nanoparticles; P0: macroscopic peritoneal dissemination; RFP: red fluorescent protein; SPION: superparamagnetic iron oxide (Fe3O4) nanoparticle.

Andrographolide Induces Noxa-Dependent Apoptosis by Transactivating ATF4 in Human Lung Adenocarcinoma Cells.

Lung adenocarcinoma is the most common pathological type of lung cancer with poor patient outcomes; therefore, developing novel therapeutic agents is critically needed. Andrographolide (AD), a major active component derived from the traditional Chinese medicine (TCM) Andrographis paniculate, is a potential antitumor drug, but the role of AD in lung adenocarcinoma remains poorly understood. In the present study, we demonstrated that AD inhibited the proliferation of broad-spectrum lung cancer cell lines in a dose-dependent manner. Meanwhile, we found that a high dose of AD induced Noxa-dependent apoptosis in human lung adenocarcinoma cells (A549 and H1299). Further studies revealed that Noxa was transcriptionally activated by activating transcription factor 4 (ATF4) in AD-induced apoptosis. Knockdown of ATF4 by small interfering RNA (siRNA) significantly diminished the transactivation of Noxa as well as the apoptotic population induced by AD. These results of the present study indicated that AD induced apoptosis of human lung adenocarcinoma cells by activating the ATF4/Noxa axis and supporting the development of AD as a promising candidate for the new era of chemotherapy.

Traditional Chinese Medicine Brucea Javanica Oil Enhances the Efficacy of Anlotinib in a Mouse Model of Liver-Metastasis of Small-cell Lung Cancer.

BACKGROUND/AIM: Small-cell lung cancer (SCLC) is a recalcitrant disease with liver and other metastasis. The present study evaluated the efficacy of the traditional Chinese medicine Brucea javanica oil (BJO) combined with anlotinib, a multi-tyrosine kinase inhibitor with anti-angiogenic activity, on a nude-mouse model of SCLC liver metastasis. MATERIALS AND METHODS: The mouse model was established by injecting NCI-H446 cells (1×106) in Matrigel (20 µl) into the upper liver lobe. All animals were randomized and assigned to three groups: Control (n=8); anlotinib alone (n=8; 3 mg/kg, qd×14+7-day interval with two cycles, oral); anlotinib plus BJO (n=8; 3 mg/kg anlotinib qd×14+7-day interval with two cycles, orally; BJO: 1 g/kg, qd×6 weeks, orally). Body weight was determined every week. Six weeks after initial treatment, tumors were collected for analysis of angiogenesis using immunohistochemistry. RESULTS: The combination of anlotinib and BJO significantly inhibited growth of SCLC liver metastases and angiogenesis more than anlotinib monotherapy (p=0.043). In addition, BJO alleviated body-weight loss associated with anlotinib therapy, including general mouse condition. CONCLUSION: The results of the present study indicate that the combination of anlotinib with BJO is promisingly active against liver metastases of SCLC, and has clinical potential.

Brucea javanica Increases Survival and Enhances Gemcitabine Efficacy in a Patient-derived Orthotopic Xenograft (PDOX) Mouse Model of Pancreatic Cancer.

BACKGROUND/AIM: Traditional Chinese medicine (TCM) Brucea javanica (BJO) has shown anti-proliferation efficacy on human carcinoma cells in vitro. The aim of the present study was to evaluate for the first time the efficacy of BJO combined with the first-line chemotherapeutic drug gemcitabine (GEM) on tumor growth-inhibition and survival in a pancreatic cancer patient-derived orthotopic xenograft (PDOX) mouse model. MATERIALS AND METHODS: The pancreatic cancer tumor fragment originated from a patient at the Hefei First People's Hospital (Anhui, PR China). The surgical specimen was transplanted orthotopically in nude mice using surgical orthotopic implantation (SOI). All mice were randomized and assigned to 5 groups: G1: saline vehicle (0.1ml per mouse, oral, once per day); G2: GEM [100 mg/kg, intraperitoneal (i.p), twice per week]; G3: GEM+BJO [100 mg/kg GEM, i.p, twice per week+1g/kg BJO, oral, once per day (qd)]; G4: BJO (1g/kg, oral, qd). Group 5 and Group 6 were used to observe survival [G5: saline vehicle (0.1ml per mouse, oral, qd), G6: BJO (1g/kg, oral, qd)]. Body weight and tumor volume were measured twice per week. TUNEL staining was used to determine apoptosis. RESULTS: The combination of GEM + BJO resulted in a reduced tumor growth rate (p<0.05) and greater apoptosis (p<0.05) compared to the vehicle control and GEM monotherapy. In addition, the BJO-treated group showed a statistically significant increase in survival compared to the vehicle control (p<0.05). CONCLUSION: BJO is a promising non-toxic TCM to effectively treat pancreatic cancer, both as monotherapy and in combination with first-line GEM therapy.

Near-infrared photoimmunotherapy is effective treatment for colorectal cancer in orthotopic nude-mouse models.

BACKGROUND: Photoimmunotherapy (PIT) employs the use of a near-infrared (NIR) laser to activate an antibody conjugated to a NIR-activatable dye to induce cancer cell death. PIT has shown to be effective in a number of studies, however, there are no data on its use in colorectal cancer in an orthotopic model. METHODS: Humanized anti-CEA antibody (M5A) was conjugated to NIR-activatable IRDye700DX (M5A-700). PIT was validated in vitro with a colon cancer cell-line, using a laser intensity of either 4 J/cm2, 8 J/cm2, or 16 J/cm2. Orthotopic colon cancer mouse models were established by surgical implantation of LS174T tumor fragments onto the cecum. M5A-700 was administered and PIT was performed 24 hours later using a 690 nm laser. Repeat PIT was performed after 7 days in one group. Control mice received laser treatment only. RESULTS: In vitro PIT demonstrated tumor cell death in a laser intensity dose-dependent fashion. In orthotopic models, control mice demonstrated persistent tumor growth. Mice that underwent PIT one time had tumor growth arrested for one week, after which re-growth occurred. The group that received repeated PIT exposure had persistent inhibition of tumor growth. CONCLUSION: PIT arrests tumor growth in colon cancer orthotopic nude-mouse models. Repeated PIT arrests colon cancer growth for a longer period of time. PIT may be a useful therapy in the future as an adjunct to surgical resection or as primary therapy to suppress tumor progression.

Combination Treatment With Sorafenib and Everolimus Regresses a Doxorubicin-resistant Osteosarcoma in a PDOX Mouse Model.

BACKGROUND/AIM: Osteosarcoma is a rare but recalcitrant type of bone cancer. To discover an effective therapy for osteosarcoma, we used a patient-derived orthotopic xenograft (PDOX) mouse model. A PDOX mouse model has been established for all major cancer types. Strong synergistic efficacy of sorafenib (SFN) and everolimus (EVL) has been demonstrated in several cancers. In the present study, we examined the efficacy of a SFN and EVL combination on a doxorubicin (DOX)-resistant osteosarcoma PDOX. MATERIALS AND METHODS: The osteosarcoma PDOX models were randomly divided into five treatment groups, each containing six mice: Control; DOX; SFN; EVL; and a combination of SFN and EVL. Mice were treated for 14 days. To observe the efficacy of these treatments, tumor size and body weight were measured, and histological sections were analyzed. RESULTS: Tumor growth regression was observed only in the mice treated with the combination of SFN-EVL. Histological analysis revealed necrosis with degenerative changes in tumors treated with a combination of SFN-EVL. CONCLUSION: A SFN-EVL combination could be a novel effective treatment option for osteosarcoma.

Traditional Chinese Medicine (TCM) Astragalus Membranaceus and Curcuma Wenyujin Promote Vascular Normalization in Tumor-derived Endothelial Cells of Human Hepatocellular Carcinoma.

BACKGROUND/AIM: The aim of the present study was to investigate the vascular normalization effect of traditional Chinese medicine Astragalus membranaceus (AM) and Curcuma wenyujin (CW) on tumor-derived endothelial cells (TECs). MATERIALS AND METHODS: TECs were isolated from the xenografted HCC cell line HepG2 expressing red fluorescent protein (RFP). The effect of AM and CW on TECs proliferation was measured using the CCK8 assay. The vascular normalization potential of AM and CW was assessed using a tube formation assay. Immunocytochemistry was performed to assess the effect of AM and CW on the expression of angiogenic maker CD34 and hypoxia-inducible factor HIF1a. RESULTS: The isolated TECs and endothelioma (EOMA) cells did not differ with regard to the expression levels of endothelial markers CD34, VEGFR-1, VEGFR-2, PDGFR-α and PDGFR-ß. All AM, CW, AM+CW and Nintedanib (Nin) showed a dose-dependent increasing inhibition effect on either TECs or EOMA cells. AM, CW and AM+CW significantly reduced HIF1a expression, increased CD34 expression and enhanced endothelial network formation in TECs or EOMA cells compared to the control. CONCLUSION: AM and CW promoted vascular normalization in tumor-derived endothelial cells of HCC, through increased expression of CD34 and reduced expression of HIF1a.

Tumor-Specific S/G2-Phase Cell Cycle Arrest of Cancer Cells by Methionine Restriction.

Cancer cells require elevated amounts of methionine (MET) and arrest their growth under conditions of MET restriction (MR). This phenomenon is termed MET dependence. Fluorescence-activated cell sorting (FACS) first indicated that the MET-dependent SV40-transformed cancer cells were arrested in the S and G2 phases of the cell cycle when under MR. This is in contrast to a G1-phase accumulation of cells, which occurs only in MET-supplemented medium at very high cell densities and which is similar to the G1 cell-cycle block which occurs in cultures of normal fibroblasts at high density. When the human PC-3 prostate carcinoma cell line was cultured in MET-free, homocysteine-containing (MET-HCY+) medium, there was an extreme increment in DNA content without cell division indicating that the cells were blocked in S phase. Recombinant methioninase (rMETase) treatment of cancer cells also selectively trapped cancer cells in S/G2: The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase trap, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S-phase-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil (5-FU) and which were highly effective in killing the cancer cells. In contrast, treatment of cancer cells with drugs in the presence of MET, only led to the majority of the cancer cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. MR has the potential for highly effective cell-cycle-based treatment strategy for cancer in the clinic.

White paper on microbial anti-cancer therapy and prevention.

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting ('Microbial Based Cancer Therapy') at the US National Cancer Institute in the summer of 2017. Here, we define 'Microbial Therapy' to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

Analysis of Anticancer Activity and Chemical Sensitization Effects of Dendropanax morbifera and Commersonia bartramia Extracts.

BACKGROUND/AIM: Dendropanax morbifera (DM) and Commersonia bartramia (CB) are possible candidates for immunotherapy. In this study, the cytotoxicity and chemical sensitization of DM and CB extracts on gynecologic and colon cancers were evaluated. MATERIALS AND METHODS: The malignant cell lines were cultured and analyzed for cytotoxicity and chemical sensitization. A mouse model was also constructed to make the condition similar to in vivo. Reverse transcription-polymerase chain reaction was conducted to determine alterations in drug-resistant genes. RESULTS: The extracts from DM and CB showed specific cytotoxicity to malignant cell lines. DM increased chemical sensitivity to cervical and ovarian cancer, while CB showed improved sensitization to endometrial cancer. The effects of the extracts were confirmed using a mouse model. The extracts induced differences in the expression levels of a number of genes related to drug resistance. CONCLUSION: DM and CB extracts could be novel agents for immunotherapy and chemical sensitization in gynecologic and colon cancers.

Ethanolic Extract of Traditional Chinese Medicine (TCM) Gamboge Inhibits Colon Cancer via the Wnt/Beta-Catenin Signaling Pathway in an Orthotopic Mouse Model.

BACKGROUND/AIM: The aim of the present study was to investigate the efficacy of an ethanolic extract of gamboge (EEG), a traditional Chinese medicine (TCM), both in vitro on colon cancer cells and in vivo in an orthotopic mouse model of human colon cancer. MATERIALS AND METHODS: The in vitro cytotoxicity of EEG on colon cancer cells was determined with the CCK8 proliferation assay and the Annexin V-PE/7-AAD apoptosis assay. Efficacy of EEG in vivo was evaluated in an orthotopic mouse model of human colon cancer implated with the green fluorescent protein-expressing human colon cancer cell line SW480-GFP. The tumor-bearing mice were treated with vehicle (0.2 ml/dose normal saline, po, daily), irinotecan (50 mg/kg/dose, ip, twice a week), 5-FU (15 mg/kg/dose, ip, every other day) as positive controls or EEG at doses of 12.5, 25 and 50 mg/kg/dose, po, daily. Real-time fluorescence imaging was performed to determine tumor inhibition in each treated group compared to the untreated controls. The protein expression of ß-catenin, MMP-7, cyclin D1 and E-cadherin in the tumors was analyzed by immunohistochemistry. RESULTS: EEG significantly induced proliferation inhibition and apoptosis of SW480 colon cancer cells in vitro in a dose-dependent manner. Tumor growth in the colon-cancer orthotopic model was significantly inhibited by irinotecan, 5-FU and all three doses of EEG. The efficacy of EEG was comparable to irinotecan and 5-FU. Irinotecan, 5-FU and 50 mg/kg EEG significantly decreased the protein expression of ß-catenin and MMP-7. Cyclin D1 expression was decreased and E-cadherin expression was increased by irinotecan, 5-FU and all three doses of EEG. CONCLUSION: The present study demonstrates anti-tumor efficacy of EEG on colon cancer both in vitro and in vivo through inducing proliferation inhibition and apoptosis of SW480 colon cancer cells and inhibiting tumor growth, respectively. EEG exerts anti-tumor activity at least partly via down-regulation of the Wnt/ß-catenin signaling pathway.