Ursolic acid sensitized colon cancer cells to chemotherapy under hypoxia by inhibiting MDR1 through HIF-1α.

OBJECTIVE: To explore the efficacy of ursolic acid in sensitizing colon cancer cells to chemotherapy under hypoxia and its underlying mechanisms. METHODS: Three colon cancer cell lines (RKO, LoVo, and SW480) were used as in vitro models. 5-Fluorouracil (5-FU) and oxaliplatin were used as chemotherapeutic drugs. Cell viability and apoptosis were tested to evaluate the sensitivity of colon cancer cells to chemotherapy. The transcription and expression levels of hypoxia-inducible factor-1α (HIF-1α), multidrug resistance gene 1 (MDR1), and vascular endothelial growth factors (VEGF) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting. Cycloheximide and MG132 were used to inhibit protein synthesis and degradation, respectively. In vitro tube formation assay was used to evaluate angiogenesis. RESULTS: We demonstrated the chemosensitizing effects of ursolic acid with 5-FU and oxaliplatin in three colon cancer cell lines under hypoxia. This effect was correlated to its inhibition of MDR1 through HIF-1α. Moreover, ursolic acid was capable of inhibiting HIF-1α accumulation with little effects on its constitutional expression in normoxia. In addition, ursolic acid also down-regulated VEGF and inhibited tumor angiogenesis. CONCLUSIONS: Ursolic acid exerted chemosensitizing effects in colon cancer cells under hypoxia by inhibiting HIF-1α accumulation and the subsequent expression of the MDR1 and VEGF.

Ursolic acid synergistically enhances the therapeutic effects of oxaliplatin in colorectal cancer.

Oxaliplatin is a key drug in chemotherapy of colorectal cancer (CRC). However, its efficacy is unsatisfied due to drug resistance of cancer cells. In this study, we tested whether a natural agent, ursolic acid, was able to enhance the efficacy of oxaliplatin for CRC. Four CRC cell lines including SW480, SW620, LoVo, and RKO were used as in vitro models, and a SW620 xenograft mouse model was used in further in vivo study. We found that ursolic acid inhibited proliferation and induced apoptosis of all four cells and enhanced the cytotoxicity of oxaliplatin. This effect was associated with down-regulation of Bcl-xL, Bcl-2, survivin, activation of caspase-3, 8, 9, and inhibition of KRAS expression and BRAF, MEK1/2, ERK1/2, p-38, JNK, AKT, IKKα, IκBα, and p65 phosphorylation of the MAPK, PI3K/AKT, and NF-κB signaling pathways. The two agents also showed synergistic effects against tumor growth in vivo. In addition, ursolic acid restored liver function and body weight of the mice treated with oxaliplatin. Thus, we concluded that ursolic acid could enhance the therapeutic effects of oxaliplatin against CRC both in vitro and in vivo, which offers an effective strategy to minimize the burden of oxaliplatin-induced adverse events and provides the groundwork for a new clinical strategy to treat CRC.

Prevalence of macro- and microvascular complications in patients with type 2 diabetes and kidney disease with or without albuminuria in a single Chinese Diabetes Centre.

OBJECTIVE: To investigate the relationship between diabetic retinopathy, neuropathy and low ankle-brachial index in mild-to-moderate chronic kidney disease of type 2 diabetic patients. METHODS: We enrolled 875 type 2 diabetic patients who were divided into two phenotypes (with or without albuminuria) and stratified into three groups (stage 1 with estimated glomerular filtration rate ⩾ 90 mL/min/1.73 m(2), stage 2 with estimated glomerular filtration rate of 60-89, stage 3 with estimated glomerular filtration rate of 30-59). The prevalence of diabetic retinopathy, neuropathy and low ankle-brachial index was compared and the risk factors of renal impairment were determined. RESULTS: Among chronic kidney disease stages, the prevalence of diabetic retinopathy increased from 42.5%, 56.6% to 66.7% in albuminuric subjects and from 29.4%, 33.0% to 50.0% with no significant trend in normoalbuminuric subjects (p = 0.005, 0.007 and 0.399 compared with albuminuric subjects in each stage). There was a significantly increased prevalence of low ankle-brachial index (17.5%, 22.6% and 44.4%) in normoalbuminuric subjects but no significant trend in albuminuric subjects. Diabetic retinopathy (odds ratio = 2.474, 95% confidence interval = 1.009-6.068) was an independent risk factor of declining kidney function in albuminuric patients. CONCLUSION: The prevalence of diabetic retinopathy was graded according to the estimated glomerular filtration rate declining in albuminuric patients while the prevalence of low ankle-brachial index was gradually increased in normoalbuminuric patients, indicating the diverse underlying mechanisms of mild to moderate chronic kidney disease between these two phenotypes.

NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

Proliferation-inhibiting and apoptosis-inducing effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitro.

OBJECTIVE: To investigate the proliferation-inhibiting and apoptosis-inducing effects of ursolic acid (UA) and oleanolic acid (OA) on multi-drug resistance (MDR) cancer cells in vitro. METHODS: UA and OA in different concentrations (0-100 µmol/L) were added separately to cultures of different cancer cell lines, including the human colon cancer cell lines SW480 and SW620, human acute myelocytic leukemia cancer cell lines HL60 and HL60/ADR, human chronic myelogenous leukemia cell lines K562 and K562/ADR, and the human breast cancer cell lines MCF-7 and MCF-7/ADR. Effects of UA and OA on cell proliferation were detected by 3-(4,5-dimethyl-2-thiazole)-2-5-biphenly-tetrazole bromide (MTT) method and effects on cell apoptosis were tested by flow cytometry (FCM) and Western blot at 24, 48, and 72 h after treatment. RESULTS: Both UA and OA showed significant inhibition on parent and MDR cell lines in a time- and concentration-dependent manner; the drug-resistant multiple of them on K562 and K562/ADR as well as on HL60 and HL60/ADR was 1; the effects of UA were better than those of OA in inhibiting cell growth of solid colonic cancer and breast cancer. After SW480 cells were treated by UA at the concentrations of 0-40 µmol/L for 48 h, FCM showed that annexin V (AV) positive cells and hypodiploid peak ratio increased along with the increase in the drug's concentrations; and Western blot found that expressions of Bcl-2, Bcl-xL and survivin decreased in a concentration-dependent manner. CONCLUSIONS: Both UA and OA have antitumor effects on cancer cells with MDR, and the optimal effect is shown by UA on colonic cancer cells. Also, UA shows cell apoptosis-inducing effect on SW480, possibly by way of down-regulating the expressions of apoptosis antagonistic proteins, Bcl-2, Bcl-xL, and survivin.

Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway.

OBJECTIVE: To investigate the effects of ursolic acid on the proliferation and apoptosis of human HT-29 colon cancer cells. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays were performed to evaluate the effects of ursolic acid on the growth and apoptosis of HT-29 cells. Western blot analysis was applied to investigate the inhibitory effects of ursolic acid on the phosphorylation of the epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), and the activity of B cell leukemia-2 (Bcl-2), B cell leukemia-xL (Bcl-xL), caspase-3, and caspase-9. RESULTS: Ursolic acid inhibited the growth of HT-29 cells in dose- and time-dependent manners. The median inhibition concentration (IC50) values for 24, 48, and 72 h treatment were 26, 20, and 18 micromol/L, respectively. The apoptotic rates of 10, 20, and 40 micromol/L ursolic acid treatments for 24 h were 5.74%, 14.49%, and 33.05%, and for 48 h were 9%, 21.39%, and 40.49%, respectively. Ursolic acid suppressed the phosphorylation of EGFR, ERK1/2, p38 MAPK, and JNK, which is well correlated with its growth inhibitory effect. 10, 20, and 40 micromol/L ursolic acid significantly inhibited the proliferation of EGF-stimulated HT-29 cells (P<0.05). Cell proliferation was most significantly inhibited when treated with 10 and 20 micromol/L ursolic acid combined with 200 nmol/L AG 1478 or 10 micromol/L U0126 (P<0.01). Besides, it also down-regulated the expression of Bcl-2 and Bcl-xL and activated caspase-3 and caspase-9. CONCLUSION: Ursolic acid induces apoptosis in HT-29 cells by suppressing the EGFR/MAPK pathway, suggesting that it may be a potent agent for the treatment of colorectal cancer.

Naturally-occurring shikonin analogues--a class of necroptotic inducers that circumvent cancer drug resistance.

We previously reported that shikonin could circumvent drug resistance mediated by P-gp, Bcl-2 and Bcl-xL, by induction of necroptosis. Here, we show that the naturally-occurring shikonin analogues (deoxyshikonin, acetylshikonin, isobutyrylshikonin, beta,beta-dimethylacrylshikonin, isovalerylshikonin, alpha-methyl-n-butylshikonin) could bypass drug resistance mediated by not only P-gp, Bcl-2, and Bcl-xL, but also two additional important drug-resistant factors MRP1 and BCRP1, by induction of necroptosis. The results strengthen the previous findings that necroptotic induction could circumvent a broad spectrum of cancer drug resistance.

Bypassing cancer drug resistance by activating multiple death pathways--a proposal from the study of circumventing cancer drug resistance by induction of necroptosis.

Cancer drug resistance is a complex, dynamic, and "elusive" system rather than merely a matter of some drug-resistant factors. Current pharmacological approaches aim to restore the efficacy of the standard chemotherapy against drug-resistant cancers via reactivating apoptosis and inhibiting drug transporters, simply because the current available anticancer drugs mostly induce apoptosis and many of them are the substrates/inducers of the drug transporters. However, since there are so many different types of defects in apoptotic pathways as well as numerous drug transporters, which could simultaneously contribute to cancer drug resistance, to succeed in the approach is theoretically possible but practically extremely difficult. To circumvent cancer drug resistance is an alternative choice. Since there are multiple death pathways with molecular mechanisms distinct from each other, we previously proposed that the barriers set up in cancer cells to avoid one pathway were not problems for another. Thus, no matter how dynamic, complex, and "elusive" the resistance occurs along one death pathway (e.g., apoptosis), the resistance would be sequestered within this pathway, and would not affect another death pathway with mechanisms distinct from the former, and vice versa, e.g., apoptotic resistant cancers can be sensitive to an induction of a nonapoptotic death. Indeed, we recently demonstrated that the cancer cells resistant to apoptotic inducers such as anthracycline antibiotics, vinca alkaloids, epipodophylotoxins, were sensitive to necroptotic inducers such as shikonin. Therefore, to bypass cancer drug resistance is principally achievable by simultaneously activating multiple death pathways using combined classes of death inducers (apoptosis, autophagy, necroptosis, etc.). Although each class of death inducers has its own action window and limit in killing cancer cells, a rationalized combination of several classes of death inducers that compliment each other would maximize their efficacy while simultaneously minimizing their weakness. Such "mixed bullets" would probably achieve a good therapeutic efficacy by bypassing cancer drug resistance.