Curcumin Improves the Tumoricidal Effect of Mitomycin C by Suppressing ABCG2 Expression in Stem Cell-Like Breast Cancer Cells

<div><p>Cancer cells with stem cell–like properties contribute to the development of resistance to chemotherapy and eventually to tumor relapses. The current study investigated the potential of curcumin to reduce breast cancer stem cell (BCSC) population for sensitizing breast cancer cells to mitomycin C (MMC) both in vitro and in vivo. Curcumin improved the sensitivity of paclitaxel, cisplatin, and doxorubicin in breast cancer cell lines MCF-7 and MDA-MB-231, as shown by the more than 2-fold decrease in the half-maximal inhibitory concentration of these chemotherapeutic agents. In addition, curcumin sensitized the BCSCs of MCF-7 and MDA-MB-231 to MMC by 5- and 15-fold, respectively. The BCSCs could not grow to the fifth generation in the presence of curcumin and MMC. MMC or curcumin alone only marginally reduced the BCSC population in the mammospheres; however, together, they reduced the BCSC population in CD44⁺CD24^−/low cells by more than 75% (29.34% to 6.86%). Curcumin sensitized BCSCs through a reduction in the expression of ATP-binding cassette (ABC) transporters ABCG2 and ABCC1. We demonstrated that fumitremorgin C, a selective ABCG2 inhibitor, reduced BCSC survival to a similar degree as curcumin did. Curcumin sensitized breast cancer cells to chemotherapeutic drugs by reducing the BCSC population mainly through a reduction in the expression of ABCG2.</p></div

Effect of verapamil and curcumin on reversal of resistance to MMC in MDA-MB-231-derived BCSCs.

<p>(A) Cell survival was observed in MDA-MB-23-BCSCs and parental MDA-MB-231. Cells were treated with different concentrations for 48 h. *<i>P</i> < 0.05 compared with untreated control. (B) Dose- and time-dependent effect of MMC with and without verapamil and curcumin. *<i>P</i> < 0.05 compared with MMC and verapamil at corresponding doses. Cell survival was analyzed using MTT. Values are mean ± SD from 3 independent experiments. (C) Western blot analysis of ABCC2, ABCB1, ABCG2, and ABCC1 by curcumin and MMC with and without verapamil treatment in MDA-MB-231 BCSCs. Cells were incubated with 40 μmol/L curcumin, 25 μmol/L MMC, 10 μM/L verapamil and their combinations during 72 h. *<i>P</i> < 0.05 compared with MMC alone. (D) MDA-MB-231-derived BCSCs were treated with 40 μmol/L curcumin and 25 μmol/L MMC alone and together with and without fumitremorgin C and probenecid for 72 h. An MTT assay was performed. Values are mean ± SD from 3 independent experiments. *<i>P</i> < 0.05 compared with MMC alone.</p

Curcumin and chemotherapeutic drugs (paclitaxol, cisplatin, and doxorubicin) alone and together inhibited the cell viability of MDA-MB-231 and MCF-7 cells.

<p>Cells were treated with different concentrations of paclitaxel, cisplatin, and doxorubicin and/or 40 μmol/L curcumin (IC₅₀). An MTT assay was used to determine cell viability at 48 h. Data are presented as means ± SD (n = 3). *<i>P</i> < 0.05 and compared with untreated control.</p

Effect of MMC or MMC+ curcumin on tumor outgrowth in MDA-MB-231 BCSCs xenografts.

<p>BALB/c nude mice bearing MDA-MB-231 BCSCs in fat pads as xenografts were treated with daily i.p. injection of control or curcumin and MMC for 4 wk. Tumor volumes (A) and mouse body weights (B) were determined as described. Data are presented as means ± SD (n = 10). *<i>P</i> < 0.05, compared with MMC alone.</p

Curcumin and MMC alone and together inhibited mammosphere formation in MCF-7 and MDA-MB-231 BCSCs.

<p>(A) MSFE of MCF-7 (a) and MDA-MB-231 (b) BCSCs treated with different doses of curcumin and MMC and their combination was calculated as the percentage of the number of mammosphere (diameter >50 μm) formed in 7 d/original number of cells seeded and is expressed as mean ± SD (n = 3). **<i>P</i> < 0.05, compared with MMC 0 μmol/L. *<i>P</i> < 0.05, compared with curcumin 5 μmol/L. #<i>P</i> < 0.05, compared with curcumin 20 μmol/L. (B) Self-renewal capacity of BCSCs was observed in curcumin 5 μmol/L and MMC 0.1 μmol/L alone and together in MCF-7 (a) and both curcumin 5 μmol/L and MMC 0.5 μmol/L alone or together in MDA-MB-231 (b). Data are presented as mean ± SD (n = 3). *<i>P</i> < 0.05, compared with control in different passages. (C) Curcumin sensitizes cancer stem cells and functions synergistically with MMC. Presented are representative flow cytometry dot plots for CD44 and CD24 cell marker expression in MDA-MB-231–derived BCSCs. a, untreated control; b, curcumin treatment; c, MMC treatment; and d, curcumin + MMC treatment.</p

Replication capacity and adaptability of a severe fever with thrombocytopenia syndrome virus at different temperatures

<div><p>Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease caused by the SFTS virus (SFTSV). Although fever and thrombocytopenia are the typical manifestations of SFTS, a specific SFTS case with no fever was observed in Zhejiang, China. In this report, we aimed to explore the probable reason for the absence of fever by analyzing the genetic characteristics and temperature sensitivity (ts) of the SFTSV strain ZJ2013-06, which was isolated from the specific case. Phylogenetically, different clusters of SFTSV strains circulated in Zhejiang. ZJ2013-06 was farthest from ZJ2014-02, an isolate belonging to a Chinese dominant cluster, and nearest to the coastal strain NB24/CHN/2013. Ts tests, performed on Vero cells at 37°C and 39°C, indicated that ZJ2013-06 had restricted replication at 39°C. Its viral loads were substantially reduced at 39°C compared with that at 37°C (approximately 100-fold reduction) and were significantly lower than that of ZJ2014-02 at 39°C (<i>P</i> < 0.01). By adaptive culture at 39°C, the induced strain ZJ2013-06-P7 was obtained. Owing to a reverse mutation (S1616), ZJ2013-06-P7 lost the ts of the original strain, displaying similar replication processes with NB24/CHN/2013. The results indicated that the amino acid residue 1616 was related to the ts characteristics of ZJ2013-06. Our study revealed that ZJ2013-06 was temperature-sensitive and may be related to the absence of fever in our case.</p></div

Replication capacities of ZJ2013-06 at different temperatures.

<p>Replication capacities of ZJ2013-06 at different temperatures.</p

Temperature sensitivity of NB24/CHN/2013 and ZJ2013-06-P7.

<p>Temperature sensitivity of NB24/CHN/2013 and ZJ2013-06-P7.</p

The real-time RT-PCR results of HEV71, CVA16, and HEVs for different types of HFMD clinical specimens.

<p>Note:</p><p>1. *Among 48 HEVs-positive specimens, one specimen was positive for HEVs, but negative for HEV71 or CVA16, which was identified as Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene.</p><p>2. For the positive specimens for HEV71 or CVA16, the results of real-time RT-PCR for HEVs were also positive.</p><p>3. A total of 85 HEV71 and 16 CVA16 were identified using RT-PCR method among the 213 HFMD clinical specimens, which were lower than those of the real-time RT-PCR assay.</p><p>4. The multiplex real-time RT-PCR assay was shown to exhibit 100% specificity in detecting HEV71 and CVA16, which was confirmed by sequencing.</p

The sequences of the primers and probe for HEV, HEV71, and CVA16.

<p>Note:</p><p>1. “a” indicates the sequence position of the primers and probes referring to strain BrCr of HEV71 (GenBank accession number U22521); “b” indicates the sequence position of the primers and probe referring to strain G-10 of CVA16 (GenBank accession number U05876).</p><p>2. The lengths of real-time RT-PCR products for HEV71, CVA16, and HEV were 76, 104, and 194 nt, respectively.</p><p>3. The primers and probes of HEV71, CVA16, and HEV have been applied for national invention patent in China, and the numbers of the patents are ZL200810063097.5, ZL200810121454.9, and ZL200810121453.4, respectively.</p