Розмарин и рак Rosmarinus officinalis & cancer Научные исследования

Discussion

In the present study we investigated the effectiveness of carnosol, a natural compound, against the TNBC MDA-MB-231 cells. We showed that carnosol efficiently inhibited in vitro and in vivo the growth of the MDA-MB-231 cells. We found that carnosol induced cell cycle arrest at G2 phase confirmed by a decrease in p(ser10) histone H3 . G2 arrest by carnosol was also shown in prostate cancer PC3 cells [17] and was associated with an upregulation of the CDK inhibitors p21 and p27. In MDA-MB-231 cells, however, we found that the cell cycle arrest correlated with an upregulation of the CDK inhibitor p21 and down regulation of p27, thus suggesting that carnosol-induced cell cycle arrest in TNBC might involves different mechanism(s) and does not require p27. Similar to carnosol, salinomycin, a monocarboxylic polyether antibiotic, was also shown to induce G2 arrest, upregulation of P21 and downregulation of p27 in MDA-MB-231 cells [31]. We also found that arrested MDA-MB-231 cells, in response to carnosol, undergoes apoptosis. This finding is in agreement with previous reports demonstrating that the anticancer effect of carnosol on leukemia [19], prostate cancer [15], [17] and colon cancer [18] cell lines was due to its apoptosis-inducing activity. Interestingly, our results showed for the first time that carnosol activated both intrinsic and extrinsic apoptotic pathway in TNBC through an activation of caspase 9 and 8 respectively. Carnosol induced expression of pro-apoptotic protein with concomitant decrease of Bcl2 expression and loss of mitochondrial membrane potential.

There is increasing number of evidences highlighting the central role of ROS production in inducing autophagy and cell death in many cancer cell types. In fact, several anticancer agents were shown to mediate their effect through ROS, and inhibition of ROS production by ROS scavenger blocks autophagy and cell death in many cancer types [34, 35, 38, and 39]. Similarly, in this study we observed carnosol-enhanced ROS generation in breast cancer cells in dose- and time-dependent manner. Recent studies showed that the extent of ROS level produced, in response to anticancer agents, elicits different responses in cancer cells. While low level of ROS was shown to induce autophagy, excessive ROS accumulation triggered both apoptosis and cell death. In agreement with these studies, we found that, depending on the concentration and time of exposure, carnosol elicited different responses in MDA-MB-231 cells. We found that carnosol at non cytotoxic concentration (25 µM) resulted in low level in ROS production and γH2AX activation, and induced autophagy. We believe that in this context, autophagy was induced to remove damaged organelles from treated cells. However, exposure of cells, for short period of time, to higher concentration of carnosol (≥50 µM) triggered autophagy as self-defense survival mechanism. A prolonged exposure to such concentration of carnosol led to excessive ROS production, which ultimately resulted in higher levels of oxidative damage that exceeds the cell's repair capabilities that eventually caused programmed cell death through activation of intrinsic and extrinsic apoptotic pathways. These findings which highlight the essential role of ROS accumulation are supported by the following evidence: The abrogation of ROS production by the ROS scavenger tiron, totally blocked autophagy and apoptosis in cells treated with 50 µM carnosol and significantly attenuated both events at a higher concentration (100 µM). Altogether, these data strongly demonstrate that ROS production in response to carnosol act as an upstream effector for autophagy and subsequent apoptosis induction. Similar mechanism has been described by Lin and collaborators for safingol, an anticancer drug in phase I clinical trial, which has been shown to mediate a concentration-dependent effect in MDA-MB-231 and HT29 cancer cells [38]. These authors showed that Low concentration of safingol triggered autophagy as damage repair mechanism, while higher concentration led to cell death [38].

DNA damage caused by genotoxic chemicals or ROS accumulation was shown to induce autophagy. Still the mechanisms by which DNA damage triggers autophagy are unclear [40]. Interestingly, we found that carnosol induced a dose-dependent increase in γH2AX, a marker of DNA damage, detected as early as 3 h, a time that coincides with autophagy induction. Most importantly, we found that inhibition of ROS production by tiron attenuated the activation of γH2AX. Bases on these finding, we demonstrate that DNA damage is a downstream response to ROS production that might contribute to induce autophagy and apoptosis in MDA-MB-231 cells.

Nowadays, much attention is directed toward the potential role of mitochondria damage in autophagy induction. The current hypothesis is that cells respond to mitochondrial damage in a graded fashion: when only a few mitochondria are damaged, autophagy takes place and the mitochondria are degraded; when more mitochondria are damaged, apoptosis is induced, and the cells die [32]. In agreement with this hypothesis, our electron micrograph data clearly revealed that carnosol affected the mitochondrial morphology in dose-dependent manner which ultimately resulted in loss of mitochondrial membrane potential. Thus, the induction of autophagy with or without subsequent apoptosis activation might reflect the extent of mitochondria damage in carnosol-treated cells.

In conclusion, our data are consistent with a model shown in figure 9, in which treatment with carnosol triggers oxidative damage to TNBC. The magnitude of damage, which depends upon the concentration of carnosol, determines the response of the cells. We propose that in the presence of large amounts of intracellular damages induced by high concentration of carnosol, MDA-MB-231 cells respond by triggering autophagy with subsequent apoptosis through an activation of both intrinsic and extrinsic pathway. In contrast, limited intracellular damage caused by low concentration of carnosol triggers only autophagy as repair mechanism. Our current study provides experimental evidence that the plant-derived carnosol can be promising candidate for triple negative breast cancer therapy.

In the present study, we investigated the effect of carnosol on the highly proliferative and invasive triple-negative MDA-MB-231 human breast cancer cells. We show for that carnosol blocked cell cycle at G2 phase and induced ROS-dependent apoptosis and beciln1-independent autophagy in MDA-MB-231 breast cancer cells.

Реферат агенты, которые разрушают опухолевые клетки и одновременно повышать host противоопухолевого иммунитета большой интерес в рак терапии. В настоящем исследовании, эффект carnosol на противоопухолевый иммунитет в Balb/c мыши модель фибросаркома оценивали. Carnosol вводили внутрибрюшинно ежедневно (по 5 или 10 мг/кг/день, 7 дней) опухоли-подшипник мышей (т.е. через 7 дней после инъекции опухолевых клеток). Другая группа опухоль-подшипник мышей, получавших 20 мг циклофосфамид/кг/сут (положительный контроль); итоговый группа получила транспортного средства (vehicle control). После первоначального измерения на день 0, Размер опухоли измеряли дважды в течение 7-дневного периода лечения. Однажды после окончательной обработки автомобиля/carnosol (т.е. в день 7), мышам их опухоли измеряется и затем были умерщвлены, чтобы разрешить их селезенки и опухоли заготовленная для изоляции, соответственно, спленоцитов и опухоль-ассоциированных лимфоцитов. Используя эти материалы, спонтанной и митоген-индуцированного высвобождения интерлейкина (IL)-4, IL-10 и интерферона (IFN)-γ, пролиферацию лимфоцитов и абсолютное число/относительное процентное соотношение селезенки и опухоль-ассоциированных Т-регуляторных (T_рег) и других Т-лимфоцитов подмножества были оценены. Результаты показали, что carnosol в обеих дозах значительно подавлял рост опухоли и приводит к истощению селезенки и опухоль-ассоциированных Т_рег ячеек. Этим и вызвана относительной (по сравнению с контролем мыши ячейку значения) уменьшается в splenocyte спонтанный/индуцибельной продукции IL-4 и IL-10 и увеличивается в Ифнγ и пролиферации клеток. Carnosol либо доза не вызвать изменения в процентные соотношения CD4⁺ или CD8⁺ лимфоцитов в селезенке или в опухоль-ассоциированных лимфоцитов популяции. Наблюдаемое увеличение Ифнγ, уменьшение IL-10 и IL-4 производства, и сокращение селезенки/опухолеассоциированных T_рег в ячейках могут быть признаки, отражающие потенциальную противоопухолевую активность carnosol. На основе выводов здесь, утверждается, что carnosol является вероятным кандидатом - после более полного токсиколого оценки - для возможного использования в качестве анти-рак терапевтической.