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Front Pharmacol
2018 Oct 04;9:1191. doi: 10.3389/fphar.2018.01191.
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Calein C, a Sesquiterpene Lactone Isolated From Calea Pinnatifida (Asteraceae), Inhibits Mitotic Progression and Induces Apoptosis in MCF-7 Cells.
Caldas LA
,
Horvath RO
,
Ferreira-Silva GÁ
,
Ferreira MJP
,
Ionta M
,
Sartorelli P
.
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Breast cancer is the most common cancer in women worldwide. Estrogen receptor-positive (ER+) breast cancer represents approximately 75% of diagnosed cases, while 15-20% of them are triple-negative (TN). Although there have been improvements in the therapeutic approach, the mortality rate remains elevated. Thus, it is necessary to identify new chemotherapeutic agents. The present study aimed to evaluate the effects of calein C, a sesquiterpene lactone isolated from Calea pinnatifida, on breast cancer cell lines MCF-7 (ER+), Hs578T (TN) and MDA-MB-231 (TN). Calein C significantly reduced the viability of all cell lines; however, MCF-7 cells were more responsive than MDA-MB-231 or Hs578T cells. Thus, the MCF-7 cell line was selected for further investigation. We demonstrated that calein C inhibited cell cycle progression in MCF-7 cells at M-phase. Increased frequency of mitosis was observed in calein C-treated samples compared to the control group, especially of the cell population in initial stages of the mitosis. These events were associated with the ability of calein C to modulate expression levels of critical regulators of mitosis progression. We observed a significant reduction in the relative mRNA abundance of PLK1 and AURKB along with a concomitant increase in CDKN1A (p21) in treated samples. In addition, calein C induced apoptosis in MCF-7 cells due to, at least in part, its ability to reduce the BCL2/BAX ratio. Therefore, our data provide evidence that calein C is an important antimitotic agent and should be considered for further in vivo investigations.
Figure 1. Calein C chemical structure isolated from Calea pinnatifida.
Figure 3. (A) Quantitative analysis of mitotic frequency determined from fluorescent cytological preparation labeling for microtubules, microfilaments and DNA. Total mitosis and mitosis subphases were quantified (P/PM, prophase and prometaphase; M, metaphase; A, anaphase; and T, telophase). Data represent mean ± SD from 3 independent experiments. *p < 0.05 and ***p < 0.001 according to ANOVA followed by Tukey's post-test. (B) Representative image of immunoblots showing the expression profile of p-Histone H3 (Ser10) and cyclin B1. Antineoplastic drug doxorubicin (DXR) was used as a positive control. (C) Fluorescent images obtained from control culture through confocal microscopy with a z-series projection. Note typical microtubule networks (green) in interphasic cells and bipolar mitotic spindles in mitotic cells. Microfilaments distribution pattern (red) are also shown. DNA was labeled with DAPI (blue). (D) Fluorescent images obtained from treated culture through confocal microscopy with a z-series projection. Multinucleated cell is shown in D1, and monopolar spindles are visualized in D2 and D3. (E) Quantitative analysis of frequent events observed in treated cultures. Data represent mean ± SD from 3 independent experiments. (F) Relative mRNA abundance of critical regulators of cell cycle determined by real-time PCR after 24 h of treatment using β-actin as a housekeeping gene for normalization. Data represent mean ± SEM from 4 independent experiments. *p < 0.05 and **p < 0.01 according to ANOVA followed by Tukey's post-test.
Figure 4. (A) Representative dot plots from the annexin V/7-AAD assay performed 24 h after treatment with calein C. Viable cells (lower left quadrants), early apoptotic cells (lower right quadrants), late apoptotic cells (upper right quadrants), and necrotic cells (upper left quadrants). Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control once it reduces ΔΨm. (B) Quantitative analysis performed on the annexin V assay data. Data represent the mean ± SD from 3 independent experiments. (C) Relative mRNA abundance of apoptotic genes determined by real-time PCR after 24 h of treatment using β-actin as a housekeeping gene. Data represent the mean ± SEM from 4 independent experiments. *p < 0.05 and **p < 0.01 according to ANOVA followed by Tukey's post-test.
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