BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored. METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence. RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity. CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.

Activation of Stimulation of Interferon Genes (STING) Signal and Cancer Immunotherapy.

Stimulator of interferon gene (STING), an intracellular receptor in the endoplasmic reticulum, could induce the production of cytokines such as type I interferon (IFN) by activating the cGAS-STING signal pathway. In recent years, activation of STING has shown great potential to enhance anti-tumor immunity and reshape the tumor microenvironment, which is expected to be used in tumor immunotherapy. A number of STING agonists have demonstrated promising biological activity and showed excellent synergistic anti-tumor effects in combination with other cancer therapies in preclinical studies and some clinical trials. The combination of STING agonists and ICI also showed a potent effect in improving anti-tumor immunity. In this review, we introduce the cGAS-STING signaling pathway and its effect in tumor immunity and discuss the recent strategies of activation of the STING signaling pathway and its research progress in tumor immunotherapy.

Therapies Targeting Immune Cells in Tumor Microenvironment for Non-Small Cell Lung Cancer.

The tumor microenvironment (TME) plays critical roles in immune modulation and tumor malignancies in the process of cancer development. Immune cells constitute a significant component of the TME and influence the migration and metastasis of tumor cells. Recently, a number of therapeutic approaches targeting immune cells have proven promising and have already been used to treat different types of cancer. In particular, PD-1 and PD-L1 inhibitors have been used in the first-line setting in non-small cell lung cancer (NSCLC) with PD-L1 expression ≥1%, as approved by the FDA. In this review, we provide an introduction to the immune cells in the TME and their efficacies, and then we discuss current immunotherapies in NSCLC and scientific research progress in this field.

Anticancer effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways.

BACKGROUND: Imperatorin (IM) is a furanocoumarin isolated from the root of Angelica dahurica, which is reported to have anticonvulsant and anticancer effects. In this study, the antiproliferative effect of IM on 9 human cancer cell lines was examined, and human hepatoma HepG2 cells were chosen as the target for preferential killing by IM. Particularly, the mechanism of IM-induced apoptosis and in vivo animal effects were also studied. METHODS: Cell viability was measured using MTT assay, and apoptosis was detected by Hoechst staining, annexin V-PI staining, and DNA laddering assay. Mitochondrial membrane potential was detected by JC-1 staining. Western blot analysis was employed to detect the expression of apoptosis-related proteins. In addition, the in vivo anticancer effect of IM was examined in nude mice bearing HepG2 cells. RESULTS: IM inhibited the proliferation of HepG2 cells through apoptosis induction in a time- and dose-dependent manner by observation of the nuclear morphology, DNA fragmentation, phosphatidylserine externalization, loss of mitochondrial membrane potential, release of cytochrome c into cytosol, and activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. As cell death could partly be prevented by the caspase-8 or caspase-9 inhibitor and was evidenced by the results of Western blot analysis, our results also suggest that IM-induced apoptosis is mediated through both death receptor and mitochondrial pathways. In the animal model, IM was found to effectively suppress tumor growth by 31.93 and 63.18% at dosages of 50 and 100 mg/kg, respectively, after treatment for 14 days. No significant weight loss or toxicity to the hosts was found. CONCLUSIONS: IM can function as a cancer suppressor by inducing apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways. Furthermore, the in vivo antitumor activities of IM are significant with negligible weight loss and damage to the host.

3,5-Dimethyl-H-furo[3,2-g]chromen-7-one as a potential anticancer drug by inducing p53-dependent apoptosis in human hepatoma HepG2 cells.

BACKGROUND/AIMS: Coumarins are natural compounds found in many plants that possess medical value by itself and its modified derivatives. METHOD: Six novel coumarin derivatives were synthesized and examined for their potential anticancer cytotoxicity. RESULT: Among the 6 derivatives, 3,5-dimethyl-(7)H-furo[3,2-g]chromen-7-one (DMFC) presented the strongest cytotoxicity against human hepatoma HepG2 cells in vitro with an IC(50) value of 8.46 ± 0.28 µM in a 48-hour treatment. Further experiments revealed that DMFC induced apoptosis in HepG2 cells through both extrinsic and intrinsic apoptotic pathways in a p53-dependent manner. Mechanistically, DMFC activated caspases 3, 8 and 9, depolarized mitochondrial membrane potential and induced cytochrome c and apoptosis-inducing factor release. DMFC-induced apoptosis was also characterized by DNA fragmentation, phosphatidylserine externalization and sub-G1 peak in DNA histograms. Moreover, both caspase 8 and 9 inhibitors suppressed the apoptosis induced by DMFC. Western blot analyses revealed that DMFC also significantly increased the expression levels of p53, Fas death receptor, Fas-associated death domain protein and proapoptotic Bcl-2 family members such as Bax, Bad and tBid, as well as decreased the levels of pro-survival members such as Bcl-2 and Bcl-xl. CONCLUSION: DMFC is potentially an effective therapeutic agent in liver cancer therapy.

Tea polyphenol EGCG inhibited colorectal-cancer-cell proliferation and migration via downregulation of STAT3.

BACKGROUND: Green tea is a popular beverage worldwide and epigallocatechin-3-gallate (EGCG) is the most bioactive polyphenol in green tea. Our study aims to investigate the anti-proliferation and anti-migration effects of EGCG against colorectal-cancer SW480, SW620, and LS411N cells, and elucidate the underlying mechanism. METHODS: The in vitro anti-proliferation and anti-migration effects of EGCG against colon-cancer cells were evaluated using MTT, scratch-wound-healing, and transwell-migration assays. The effects of EGCG on apoptosis were assessed by Annexin V-FITC/PI double staining and JC-1 staining. Besides, Western blotting was employed to detect the protein-expression level and elucidate the underlying pathways. Real-time qPCR and dual-luciferase reporter assay were adopted to determine the mRNA level and promoter activity. RESULTS: Our results demonstrated that treatment with EGCG resulted in significant inhibition of cell proliferation by the induction of apoptosis. EGCG also inhibited SW480 cell migration in a dose-dependent manner as assessed by wound-healing and transwell-migration assays. Western blot confirmed that EGCG induced apoptosis by the activation of Caspase-3 and PARP. In addition, both STAT3 and phosphorylated STAT3 (p-STAT3) were downregulated significantly by EGCG in three selected colorectal-cancer cell lines. EGCG treatment also resulted in a significant decrease in Bcl-2, MCL-1, and Vimentin, and an increase in E-cadherin. When STAT3 was inhibited, EGCG showed no obvious effect on cell proliferation and migration. Further investigation by luciferase-reporter-activity assay showed that EGCG suppressed the promoter activity of STAT3 and downregulated the transcription of STAT3. CONCLUSION: Our study presents evidence on the anti-proliferation and anti-migration effects of EGCG against colorectal-cancer SW480, SW620, and LS411N cells by downregulating the expression of STAT3 and suggests that EGCG could be an effective and natural supplement for colon-cancer treatment.

EGCG Enhanced the Anti-tumor Effect of Doxorubicine in Bladder Cancer via NF-κB/MDM2/p53 Pathway.

Doxorubicin (DOX), the first-line chemotherapy for bladder cancer, usually induces side effects. We previously demonstrated that green tea polyphenol EGCG had potent anti-tumor effect in bladder cancer via down regulation of NF-κB. This study aimed to investigate the additive/synergistic effect EGCG and DOX against bladder cancer. Our results demonstrated that the combined use of DOX and EGCG inhibited T24 and SW780 cell proliferation. EGCG enhanced the apoptosis induction effect of DOX in both SW780 and T24 cells and resulted in significant differences. Besides, EGCG promoted the inhibitory effect of DOX against bladder cancer cell migration. In addition, the in vivo results demonstrated that DOX in combination with EGCG showed the most potent anti-tumor effects among DOX, EGCG and DOX+EGCG treatment groups. Further mechanistic studies determined that the combination of DOX and EGCG inhibited phosphorylated NF-κB and MDM2 expression, and up-regulated p53 expression in tumor, as assessed by western blot and immunohistochemistry. Western blot in SW780 cells also confirmed that the combined use of EGCG and DOX caused significant increase in p53, p21, and cleaved-PARP expression, and induced significant inhibition in phosphorylated NF-κB and MDM2. When NF-κB was inhibited, the expression of p53 and p-MDM2 were changed, and the combination of DOX and EGCG showed no obvious effect in transwell migration and cell viability. In conclusion, the novel application of chemotherapy DOX and EGCG demonstrated potent anti-tumor, anti-migration and anti-proliferation effects against bladder cancer. EGCG enhanced the anti-tumor effect of DOX in bladder cancer via NF-κB/MDM2/p53 pathway, suggesting the potential clinical application against bladder cancer patients.

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (<10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% ± 1.2%, 94.4% ± 1.1%, and 92.4% ± 1.7% (mean ± SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway.

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. We previously found that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9. This study demonstrated that EGCG inhibited bladder cancer T24 and 5637 cell proliferation and migration via PI3K/AKT pathway, without modulation of NF-κB. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder SV-HUC-1 cells. EGCG also inhibited 5637 and T24 cell migration and invasion at 25-100 µM. Western blot confirmed that EGCG induced apoptosis in T24 and 5637cells by activation of caspases-3 and PARP. Besides, EGCG up-regulated PTEN and decreased the expression of phosphorylated PI3K, AKT in both T24 and 5637 cells. In addition, animal study demonstrated that EGCG (100 mg/kg, i.p. injected daily for 4 weeks) decreased the tumor weight in mice bearing T24 tumors by 51.2%, as compared with the untreated control. EGCG also decreased the expression of phosphorylated PI3K and AKT in tumor, indicating the important role of PI3K/AKT in EGCG inhibited tumor growth. When AKT was inhibited, EGCG showed no obvious effect in cell migration in T24 and 5637 cells. In conclusion, our study elucidated that EGCG was effective in inhibition of T24 and 5637 cell proliferation and migration, and presented evidence that EGCG inhibited cell proliferation and tumor growth by modulation of PI3K/AKT pathway.

TBK1 Promote Bladder Cancer Cell Proliferation and Migration via Akt Signaling.

Bladder cancer is a challenging and fatal malignancy and the improvement in prognosis is limited over years. Deep understanding the mechanism of bladder cancer tumorigenesis and progression will help to discover novel and effective treatment strategies. In this study, we identify non-canonical IkB kinase TBK1 is up-regulated in bladder cancer tissue and cell lines. Knockdown of TBK1 markedly inhibits cell proliferation and migration. Inhibition of TBK1 kinase activity by BX795 significantly attenuates bladder cancer cell proliferation and migration. Mechanistic study shows that overexpression of TBK1 promoted the phosphorylation of Akt, whereas knockdown of TBK1 reverses this action. Taken together, our data suggest that TBK1 modulates the malignant behaviors of bladder cancer cell via Akt signaling, revealing new insights in discovering new therapy target for bladder cancer.

BANCR: a novel oncogenic long non-coding RNA in human cancers.

Long non-coding RNAs account for large proportion of non-coding transcripts in human genomes. Though they lack of open reading framework and cannot encode protein, they can control endogenous gene expression though regulating cell life activities. They serve as transcriptional modulator, posttranscriptional processor, chromatin remodeler and splicing regulator during the process of gene modification. Moreover, long non-coding RNAs were regarded as potential tumor markers for cancer diagnosis and prognosis. BANCR was identified as a cancer-promoting long non-coding RNA in melanoma tissues. Since then, increasing studies about BANCR in cancer progression were reported. BANCR was dysregulated in various cancers including melanoma, colorectal cancer, retinoblastoma, lung carcinoma and hepatocellular carcinoma, and increased BANCR expression cause poor prognosis and shorter survival rate of cancer patients. Furthermore, the functions and mechanisms of BANCR in cancer cells have been clarified. Here, we focus on the current research on the role of BANCR in the clinical management, progression and molecular mechanisms in human cancer.

EGCG inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9.

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. Our study aims to investigate the antiproliferation and antimigration effects of EGCG against bladder cancer SW780 cells both in vitro and in vivo. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder epithelium SV-HUC-1 cells. EGCG also inhibited SW780 cell migration and invasion at 25-100 µM. Western blot confirmed that EGCG induced apoptosis in SW780 cells by activation of caspases-8, -9 and -3, Bax, Bcl-2 and PARP. Besides, animal study demonstrated that EGCG [100 mg/kg, intraperitoneal (i.p.) injection daily for 3 weeks] decreased the tumor volume significantly in mice bearing SW780 tumors, as well as the tumor weight (decreased by 68.4%). In addition, EGCG down-regulated the expression of nuclear factor-kappa B (NF-κB) and matrix metalloproteinase (MMP)-9 in both protein and mRNA level in tumor and SW780 cells. When NF-κB was inhibited, EGCG showed no obvious effect in cell proliferation and migration. In conclusion, our study demonstrated that EGCG was effective in inhibition SW780 cell proliferation and migration, and presented first evidence that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9.

Evaluation of the combined use of metronomic zoledronic acid and Coriolus versicolor in intratibial breast cancer mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor (CV) is a mushroom traditionally used for strengthening the immune system and nowadays used as immunomodulatory adjuvant in anticancer therapy. Breast cancer usually metastasizes to the skeleton, interrupts the normal bone remodeling process and causes osteolytic bone lesions. The aims of the present study were to evaluate its herb-drug interaction with metronomic zoledronate in preventing cancer propagation, metastasis and bone destruction. MATERIALS AND METHODS: Mice inoculated with human breast cancer cells tagged with a luciferase (MDA-MB-231-TXSA) in tibia were treated with CV aqueous extract, mZOL, or the combination of both for 4 weeks. Alteration of the luciferase signals in tibia, liver and lung were quantified using the IVIS imaging system. The skeletal response was evaluated using micro-computed tomography (micro-CT). In vitro experiments were carried out to confirm the in vivo findings. RESULTS: Results showed that combination of CV and mZOL diminished tumor growth without increasing the incidence of lung and liver metastasis in intratibial breast tumor model. The combination therapy also reserved the integrity of bones. In vitro studies demonstrated that combined use of CV and mZOL inhibited cancer cell proliferation and osteoclastogenesis. CONCLUSIONS: These findings suggested that combination treatment of CV and mZOL attenuated breast tumor propagation, protected against osteolytic bone lesion without significant metastases. This study provides scientific evidences on the beneficial outcome of using CV together with mZOL in the management of breast cancer and metastasis, which may lead to the development of CV as adjuvant health supplement for the control of breast cancer.

The combined use of Camellia sinensis and metronomic zoledronic acid in a breast cancer-induced osteolysis mouse model.

PURPOSE: In previous studies, we demonstrated that green tea (Camellia sinensis, CS) water extract had potent anti-tumor and anti-metastasis effects in the 4T1 mouse breast cancer xenograft model, and the metronomic regimen (0.0125 mg/kg twice a week for 4 weeks) of zoledronic acid (ZOL) was also effective in decreasing tumor burden and metastasis when compared with the conventional regimen. This study aimed to investigate the combined use of CS water extract and metronomic ZOL against tumor metastasis and bone destruction in MDA-MB-231-TXSA human breast cancer. METHODS: Female nude mice were injected with MDA-MB-231-TXSA cells into the marrow space of tibia and were treated with CS water extract and/or metronomic ZOL for 4 weeks. Tumor growth and metastasis to lungs and livers were assessed by in vivo bioluminescence imaging. Abilities of migration and invasion of MDA-MB-231-TXSA cells were also evaluated in vitro. RESULTS: Our results demonstrated that combination of CS and ZOL had the most potent effects on tumor burden and metastasis to bone, lung and liver, while treatment with CS or ZOL alone significantly protected the bone from cancer-induced osteolysis. In vitro, the combined use of CS + ZOL significantly inhibited MDA-MB-231-TXSA cell migration and invasion. Mechanistic zymography studies showed that the enzyme activities of MMP-9 and MMP-2 were significantly suppressed by CS and CS + ZOL. CONCLUSIONS: The combination of CS plus metronomic ZOL demonstrated potent anti-tumor, anti-metastasis and anti-osteolysis effects against breast cancer, suggesting the potential clinical application against breast cancer patients.

The combined use of Camellia sinensis and metronomic zoledronate in 4T1 mouse carcinoma against tumor growth and metastasis.

In previous studies, we demonstrated that the green tea Camellia sinensis (CS) water extract had potent antitumor and antimetastatic effects on 4T1 breast cancer. The metronomic regimen (0.0125 mg/kg twice a week for 4 weeks) of zoledronate (ZOL) was found to be effective in decreasing tumor burden and metastasis as compared with conventional regimen. The aim of the present study was to investigate the antitumor, antimetastatic and anti-osteolytic effects of the combined use of CS water extract and metronomic ZOL against 4T1 breast carcinoma in vitro and in vivo. The results demonstrated that the combination of CS+ZOL exerted a more potent effect on lung and liver by decreasing tumor burden and metastasis, when compared to CS or metronomic ZOL as monotherapies. The combination of CS+ZOL demonstrated optimal bone protection against breast cancer-induced osteolysis for the three groups of CS, ZOL and CS+ZOL. The in vitro results further demonstrated that ZOL enhanced CS-induced apoptosis in 4T1 cells as assessed by the Annexin V-FITC/PI staining and caspase-3 activity assays. In addition, the combined use of CS+ZOL significantly inhibited 4T1 cell migration. Mechanistic studies showed that the enzyme levels of matrix metalloproteinases (MMP)-2 and MMP-9 were suppressed significantly by CS+ZOL. In conclusion, to the best of our knowledge, this is the first study to investigate the novel combined application of herbal extract CS and chemotherapy ZOL in 4T1 breast cancer. The combination of CS plus metronomic ZOL demonstrated significant antitumor, antimetastatic and anti-osteolytic effects against breast cancer, and suggested potential clinical application for breast cancer patients.

Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice.

Green tea (Camellia sinensis, CS), a kind of Chinese tea commonly consumed as a healthy beverage, has been demonstrated to have various biological activities, including antioxidation, antiobesity and anticancer. Our study aims to investigate the antitumor, antimetastasis and antiosteolytic effects of CS aqueous extract both in vitro and in vivo using metastasis-specific mouse mammary carcinoma 4T1 cells. Our results showed that treatment of 4T1 cells with CS aqueous extract resulted in significant inhibition of 4T1 cell proliferation. CS extract induced 4T1 apoptosis in a dose-dependent manner as assessed by annexin-V and propidium iodide staining and caspase-3 activity. Western blot analysis showed that CS increased the expression of Bax-to-Bcl-2 ratio and activated caspase-8 and caspase-3 to induce apoptosis. CS also inhibited 4T1 cell migration and invasion at 0.06-0.125 mg/ml. In addition, CS extract (0.6 g/kg, orally fed daily for 4 weeks) was effective in decreasing the tumor weight by 34.8% in female BALB/c mice against water treatment control (100%). Apart from the antitumor effect, CS extract significantly decreased lung and liver metastasis in BALB/c mice bearing 4T1 tumors by 54.5% and 72.6%, respectively. Furthermore, micro-computed tomography and in vitro osteoclast staining analysis suggested that CS extract was effective in bone protection against breast cancer-induced bone destruction. In conclusion, the present study demonstrated that the CS aqueous extract, which closely mimics green tea beverage, has potent antitumor and antimetastasis effects in breast cancer and could protect the bone from breast cancer-induced bone destruction.

In vivo and in vitro anti-tumor and anti-metastasis effects of Coriolus versicolor aqueous extract on mouse mammary 4T1 carcinoma.

Coriolus versicolor (CV), a medicinal mushroom widely consumed in Asian countries, has been demonstrated to be effective in stimulation of immune system and inhibition of tumor growth. The present study aimed to investigate the anti-tumor and anti-metastasis effects of CV aqueous extract in mouse mammary carcinoma 4T1 cells and in 4T1-tumor bearing mouse model. Our results showed that CV aqueous extract (0.125-2 mg/ml) did not inhibit 4T1 cell proliferation while the non-cytotoxic dose of CV extract (1-2 mg/ml) significantly inhibited cell migration and invasion (p<0.05). Besides, the enzyme activities and protein levels of MMP-9 were suppressed by CV extract significantly. Animal studies showed that CV aqueous extract (1 g/kg, orally-fed daily for 4 weeks) was effective in decreasing the tumor weight by 36%, and decreased the lung metastasis by 70.8% against untreated control. Besides, micro-CT analysis of the tumor-bearing mice tibias indicated that CV extract was effective in bone protection against breast cancer-induced bone destruction as the bone volume was significantly increased. On the other hand, CV aqueous extract treatments resulted in remarkable immunomodulatory effects, which was reflected by the augmentation of IL-2, 6, 12, TNF-α and IFN-γ productions from the spleen lymphocytes of CV-treated tumor-bearing mice. In conclusion, our results demonstrated for the first time that the CV aqueous extract exhibited anti-tumor, anti-metastasis and immunomodulation effects in metastatic breast cancer mouse model, and could protect the bone from breast cancer-induced bone destruction. These findings provided scientific evidences for the clinical application of CV aqueous extract in breast cancer patients.

Pharmacologic inhibition of bone resorption prevents cancer-induced osteolysis but enhances soft tissue metastasis in a mouse model of osteolytic breast cancer.

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

Structure elucidation and immunomodulatory activity of a beta glucan from the fruiting bodies of Ganoderma sinense.

A polysaccharide named GSP-2 with a molecular size of 32 kDa was isolated from the fruiting bodies of Ganoderma sinense. Its structure was well elucidated, by a combined utilization of chemical and spectroscopic techniques, to be a ß-glucan with a backbone of (1→4)- and (1→6)-Glcp, bearing terminal- and (1→3)-Glcp side-chains at O-3 position of (1→6)-Glcp. Immunological assay exhibited that GSP-2 significantly induced the proliferation of BALB/c mice splenocytes with target on only B cells, and enhanced the production of several cytokines in human peripheral blood mononuclear cells and derived dendritic cells. Besides, the fluorescent labeled GSP-2 was phagocytosed by the RAW 264.7 cells and induced the nitric oxide secretion from the cells.

Anti-tumor and anti-osteolysis effects of the metronomic use of zoledronic acid in primary and metastatic breast cancer mouse models.

This study aims to determine the effect of metronomic (0.0125 mg/kg twice a week for 4 weeks) zoledronic acid (ZOL) on cancer propagation and osteolysis against both metastatic and primary breast cancer in mice model. From our results, metronomic ZOL resulted in a significant reduction of tumor burden and did not promote lung or liver metastasis. The metronomic ZOL appeared to be more effective than the conventional regimen (0.1 mg/kg once in 4 weeks) in reducing breast cancer tumor burden, and regulating its movement to lung and liver. This dosing schedule of ZOL showed great potential against metastatic breast cancer.