Brazilein, a compound isolated from Caesalpinia sappan Linn., induced growth inhibition in breast cancer cells via involvement of GSK-3ß/ß-Catenin/cyclin D1 pathway.

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC50 value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 µmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3ß and following reduced ß-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3ß/ß-Catenin pathway was involved in its action mechanism.

Secalonic acid D reduced the percentage of side populations by down-regulating the expression of ABCG2.

The side population cells characterized by the ability to transport Hoechst 33342 out of cells have been identified as cancer stem-like cells. ABCG2 is found to confer the side population (SP) phenotype, multidrug resistance (MDR) and tumor recurrence. In this study, we found secalonic acid D (SAD), a metabolite of marine-derived mangrove endophytic fungus, showed potent anticancer effect on ABCB1-, ABCC1- and ABCG2- overexpressing multidrug resistance cells by MTT assay. Furthermore, SAD could down-regulate the expression of ABCG2 and decrease the percentage of SP cells in lung cancer cells. The MTT assay showed the sorted SP cells were sensitive to SAD and we also found SAD could inhibit the sphere-forming ability of SP cells. Although SAD did not affect the expression of ABCG2 mRNA, it shortened the half-life of ABCG2 protein by activating calpain 1. These results implicated that SAD could be developed as a leading compound to target cancer stem cells and would be a promising agent to treat lung cancer patients.

Euphorbia factor L1 reverses ABCB1-mediated multidrug resistance involving interaction with ABCB1 independent of ABCB1 downregualtion.

Euphorbia factor L1 (EFL1) belongs to diterpenoids of genus Euphorbia. In this article, its reversal activity against ABCB1-mediated MDR in KBv200 and MCF-7/adr cells was reported. However, EFL1 did not alter the sensitivity of KB and MCF-7 cells to chemotherapeutic agents. Meanwhile, EFL1 significantly increased accumulation of doxorubicin and rhodamine 123 in KBv200 and MCF-7/adr cells, showing no significant influence on that of KB and MCF-7 cells. Furthermore, EFL1 could enhance the ATP hydrolysis activity of ABCB1 stimulated by verapamil. At the same time, EFL1 inhibited the efflux of ABCB1 in KBv200 and MCF-7/adr cells. In addition, EFL1 did not downregulate expression of ABCB1 in KBv200 and MCF-7/adr cells either in mRNA or protein level.

Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters.

Apatinib, a small-molecule multitargeted tyrosine kinase inhibitor, is in phase III clinical trial for the treatment of patients with non-small-cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr, and HEK293/ABCB1 cells overexpressing ABCB1 and in S1-M1-80, MCF-7/FLV1000, and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photoaffinity labeling of both ABCB1 and ABCG2 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration that produced a reversal of MDR, did not significantly alter the ABCB1 or ABCG2 protein or mRNA expression levels or the phosphorylation of AKT and extracellular signal-regulated kinase 1/2 (ERK1/2). Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1-resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking the AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs.

Anticancer effect and structure-activity analysis of marine products isolated from metabolites of mangrove fungi in the South China Sea.

Marine-derived fungi provide plenty of structurally unique and biologically active secondary metabolites. We screened 87 marine products from mangrove fungi in the South China Sea for anticancer activity by MTT assay. 14% of the compounds (11/86) exhibited a potent activity against cancer in vitro. Importantly, some compounds such as compounds 78 and 81 appeared to be promising for treating cancer patients with multidrug resistance, which should encourage more efforts to isolate promising candidates for further development as clinically useful chemotherapeutic drugs. Furthermore, DNA intercalation was not involved in their anticancer activities, as determined by DNA binding assay. On the other hand, the structure-activity analysis indicated that the hydroxyl group was important for their cytotoxic activity and that bulky functional groups such as phenyl rings could result in a loss of biological activity, which will direct the further development of marine product-based derivatives.

Anthracenedione derivatives as anticancer agents isolated from secondary metabolites of the mangrove endophytic fungi.

In this article, we report anticancer activity of 14 anthracenedione derivatives separated from the secondary metabolites of the mangrove endophytic fungi Halorosellinia sp. (No. 1403) and Guignardia sp. (No. 4382). Some of them inhibited potently the growth of KB and KBv200 cells, among which compound 6 displayed strong cytotoxicity with IC(50) values of 3.17 and 3.21 microM to KB and KBv200 cells, respectively. Furthermore, we demonstrate that the mechanism involved in the apoptosis induced by compound 6 is probably related to mitochondrial dysfunction. Additionally, the structure-activity relationships of these compounds are discussed.

Reversal of P-gp-mediated multidrug resistance by Bromotetrandrine in vivo is associated with enhanced accumulation of chemotherapeutical drug in tumor tissue.

BACKGROUND: Our previous studies have shown that tetrandrine (Tet) reverses the effect of P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) both in vitro and in vivo. 5-Bromotetrandrine (Br-Tet) is a newly synthesized brominated derivative of Tet. In this study, we investigated the MDR reversal activity of Br-Tet in vitro and in vivo and the mechanism involved in this reversal. MATERIALS AND METHODS: The ability of Br-Tet to reverse drug resistance in vitro was evaluated by MTT assay in human MDR KBv200 cells and the parental drug-sensitive KB cells. A KBv200 cell xenograft model was established to investigate the MDR reversal activity of Br-Tet in vivo. Doxorubicin (Dox) accumulation in KBv200 and KB cell lines was determined by flow cytometry and Dox accumulation in KBv200 xenografts tissue was examined by spectrofluorometer. The effect of Br-Tet on the expression of P-glycoprotein was detected by flow cytometry and Western blot, respectively. RESULTS: Br-Tet significantly enhanced the cytotoxicity of Dox, paclitaxel, taxotere, vincristine and epirubicin in KBv200 cells but not in KB cells. Co-administration of 10 mg/kg Br-Tet and 2 mg/kg epirubicin significantly enhanced the antitumor activity of epirubicin without increasing the toxicity. Br-Tet increased the Dox accumulation in the MDR KBv200 cell line and in KBv200 xenograft tissue in a time- and dose-dependent manner. However, it did not reduce the expression of P-gp in KBv200 cells. CONCLUSION: Br-Tet caused a significant reversal of P-gp-mediated MDR, not only in vitro but also in vivo. The MDR reversal activity of Br-Tet in vivo was associated with the enhancement of accumulation of chemotherapeutical drugs in tumor tissue.

[Impact of meticulousness of pathologists on lymph node harvest after radical resection of invasive rectal carcinoma].

OBJECTIVE: To analyze the impact of meticulousness of pathologists on the lymph node harvest after radical resection of invasive rectal carcinoma. METHODS: From January 2008 to May 2009, the clinical data of rectal cancer patients undergone operation were reviewed retrospectively. After multidisciplinary cooperation on rectal cancer, a new rule was applied to request the pathologists to find no less than 15 nodes in single colorectal specimen from January 2009. Patients were divided into two groups (2008 group and 2009 group) and the node harvest numbers were compared. Excluded criteria were recurrent colorectal tumor, Tis tumor, R(1) or R(2) resection, tumor resection transanally or endoscopically, the cases enrolled in other prospective research, synchronous diseases affecting the surgical procedure for the rectal cancer (familial adenomatous polyposis, synchronous colorectal carcinoma) and rectal cancer receiving neoadjuvant chemoradiation. Statistical analysis was performed using One-Sample Kolmogorov- Smirnov test, Mann-Whitney test, Independent-Samples T test and Chi-Square test(SPSS 15.0). RESULTS: A total of 232 patients were identified, including 76 cases in the 2009 group and 156 cases in 2008 group. The lymph node retrieval in the 2009 group was significantly more than that in 2008 group (16.0+/-0.3 vs 11.4+/-0.3, P<0.01). A significantly higher percentage of patients was found in 2009 group with a lymph node harvest equal to or more than 12 nodes (72/76 vs 71/156, P<0.01). There were no significant differences in gender (46/76 vs 86/156, P=0.436), age (58.1+/-1.3 vs 59.2+/-1.1, P=0.527), distance from tumor to anal verge (7.4+/-0.4 vs 7.1+/-0.3, P=0.761), proportion of sphincter-sparing surgery (67/76 vs 140/156, P=0.715), ratio of well and moderate differentiated tumors (68/76 vs 125/156, P=0.074) and overall TNM stage (P=0.167) between the two groups. CONCLUSIONS: The lymph node harvest in 2009 group is significantly more than that in 2008 group. The good performance of pathologists could produce adequate number of lymph nodes for rectal cancer without neoadjuvant chemoradiation.

Secalonic acid D induced leukemia cell apoptosis and cell cycle arrest of G(1) with involvement of GSK-3beta/beta-catenin/c-Myc pathway.

The anticancer activities of secalonic acid D separated from the secondary metabolites of the mangrove endophytic fungus No. ZSU44 were investigated in this study. Secalonic acid D showed potent cytotoxicity to HL60 and K562 cells, and the IC(50) values were 0.38 and 0.43 mumol/L, respectively. Annexin V-FITC/PI assay and western blot indicated that secalonic acid D induced apoptosis in HL60 and K562 cells. In addition, secalonic acid D led to cell cycle arrest of G(1) phase related to downregulation of c-Myc. Moreover, our data indicated that downregulation of c-Myc and cell cycle arrest of G(1) phase were caused not by formation of G-quadruplex structures but by activation of GSK-3beta followed by degradation of beta-catenin.

Vandetanib (Zactima, ZD6474) antagonizes ABCC1- and ABCG2-mediated multidrug resistance by inhibition of their transport function.

BACKGROUND: ABCC1 and ABCG2 are ubiquitous ATP-binding cassette transmembrane proteins that play an important role in multidrug resistance (MDR). In this study, we evaluated the possible interaction of vandetanib, an orally administered drug inhibiting multiple receptor tyrosine kinases, with ABCC1 and ABCG2 in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: MDR cancer cells overexpressing ABCC1 or ABCG2 and their sensitive parental cell lines were used. MTT assay showed that vandetanib had moderate and almost equal-potent anti-proliferative activity in both sensitive parental and MDR cancer cells. Concomitant treatment of MDR cells with vandetanib and specific inhibitors of ABCC1 or ABCG2 did not alter their sensitivity to the former drug. On the other hand, clinically attainable but non-toxic doses of vandetanib were found to significantly enhance the sensitivity of MDR cancer cells to ABCC1 or ABCG2 substrate antitumor drugs. Flow cytometric analysis showed that vandetanib treatment significantly increase the intracellular accumulation of doxorubicin and rhodamine 123, substrates of ABCC1 and ABCG2 respectively, in a dose-dependent manner (P<0.05). However, no significant effect was shown in sensitive parental cell lines. Reverse transcription-PCR and Western blot analysis showed that vandetanib did not change the expression of ABCC1 and ABCG2 at both mRNA and protein levels. Furthermore, total and phosphorylated forms of AKT and ERK1/2 remained unchanged after vandetanib treatment in both sensitive and MDR cancer cells. CONCLUSIONS: Vandetanib is unlikely to be a substrate of ABCC1 or ABCG2. It overcomes ABCC1- and ABCG2-mediated drug resistance by inhibiting the transporter activity, independent of the blockade of AKT and ERK1/2 signal transduction pathways.

Cediranib (recentin, AZD2171) reverses ABCB1- and ABCC1-mediated multidrug resistance by inhibition of their transport function.

PURPOSE: Cediranib (recentin, AZD2171) is an oral small-molecule multiple receptor tyrosine kinases inhibitor. Here we investigate the ability of cediranib to reverse tumor multidrug resistance (MDR) due to overexpression of ABCB1 (P-glycoprotein) and ABCC1 (MRP1) transporters. METHODS: KBv200,MCF-7/adr, C-A120 and their parental sensitive cell lines KB, MCF-7 and KB-3-1 were used for reversal study. The intracellular accumulations of doxorubicin and rhodamine 123 were determined by flow cytometry. The expressions levels of ABCB1 and ABCC1 were investigated by Western blot and RT-PCR analyses. ATPase activity assay were performed by Luminescence. The functions of ERK in MCF-7/adr were investigated by RNA interference. RESULTS: Cediranib significantly enhanced the sensitivity of ABCB1 or ABCC1 substrates in MDR cells, with no effect found on sensitive cells. However, the expressions of these transporters were not affected and the reversal activity of cediranib was not related to the phosphorylation of AKT or ERK1/2. Further studies showed that cediranib inhibited ATPase activity of ABCB1 (P-glycoprotein) in a dose-dependent manner. CONCLUSIONS: Cediranib reverses ABCB1- and ABCC1-mediated MDR by directly inhibiting their drug efflux function. These findings may be useful for cancer combinational therapy with cediranib in the clinic.

[Study on the dynamic changing profiles of telomeric restricted fragment length among sex balanced different age groups].

OBJECTIVE: In human, both in vivo and in vitro, telomere shortening appears to be a major component of cell senescence and aging. The detailed telomere shortening status and mechanism in peripheral blood cell is needed to be further characterized. METHODS: One hundred and twenty three peripheral blood samples were collected from healthy individuals of different age groups and the mean telomeric restricted fragment (TRF) was measured using Southern Blotting with Dig labeled probe. The samples of different groups were homogenized in sex components as indicated by chi 2 test of sex ratio of different test groups (P > 0.05). RESULTS: The average length of TRF is shortening with aging and distinguished shortening dynamic profiles could be observed. Further analysis showed that there might be a shortening peak near the age of 5. CONCLUSION: There are distinguished dynamics profiles of telomere shortening among different age groups. Thus, the results indicate that it might be possible to infer individual age by telomeric restricted fragment length assay.