Plant extracts are widely known as the promising source for drug discovery, which plays a crucial role in discovering and designing novel chemotherapeutic agents [28, 29]. As a natural flavonoid derived from plants, protoapigenone, with a specific nonaromatic B-ring, exhibits remarkable cytotoxic activity, which has been served as the leading agent for developing novel anticancer drugs. On account of the structure of protoapigenone and its relevant flavonoids, we have successfully designed and synthesized the novel compound RY10-4 [14]. Interestingly, RY10-4 displayed enhanced cytotoxicity in cancer cells and decreased cytotoxicity in normal cells, which was consistent with the results in Fig. 2. However, despite its definite antitumor effect, the scientific research focused on the underlying mechanism is still limited.
In the present study, we evaluated the anti-tumor activity of RY10-4 in breast cancer. The results showed that RY10-4 effectively inhibited proliferation and induced apoptosis of MDA-MB-231, MCF-7 and SKBR-3 cells. Furthermore, xenograft assay was performed to determine the in vivo effects of RY10-4 on breast cancer, yielding similar results. Briefly, these results revealed a concentration-dependent inhibition effect of RY10-4 on breast cancer, indicating its potential in clinical application.
Ca2+ is a major second messenger in cellular signaling regulating muscle contraction, neuronal excitability, cell migration, and proliferation [16]. In order to maintain a delicate regulation of Ca2+ homeostasis, various plasma membrane and organellar Ca2+ channels, exchangers and transporters are needed. The majority of intracellular Ca2+ is stored in the endoplasmic or sarcoplasmic reticulum (ER/SR) [30]. However, it is also known that the dynamic organelles such as the mitochondria can play a major role in buffering and shaping the cytosolic Ca2+ [31, 32]. The influx of Ca2+ into mitochondria is primarily mediated by MCU, which is a mitochondrial Ca2+ channel and plays a fundamental role in mediating global calcium signaling. The transport of Ca2+ cations into the mitochondrial matrix is vital for the mitochondrial function, such as the tricarboxylic acid cycle (TCA), adenosine triphosphate (ATP) production, and ROS generation [24]. However, excessive [Ca2+]m is also able to determine cell fate, since the overload of mitochondrial Ca2+ can elevate mtROS, induce ΔΨm depolarization and promote mPTP opening, which subsequently activate the mitochondrial apoptosis. Indeed, we found the overload of [Ca2+]c and [Ca2+]m in RY10-4 treated breast cancer cells, which further disrupted the mitochondrial functions (characterized by mtROS overproduction, ΔΨm depolarization and mPTP opening as revealed in Fig. 5) and induced cell apoptosis. To clarify the process of mitochondrial Ca2+ influx, the MCU inhibitor Ru360 was administrated to the cell culture, which subsequently relieved the [Ca2+]m overload, improved the mitochondrial function and inhibited the RY10-4 induced apoptosis. These results confirmed the core role of Ca2+ homeostasis and mitochondrial function in the antitumor activity of RY10-4.
The ability to inhibit apoptosis and resist cell death is one of the well-established hallmarks of cancer [33]. Ca2+ and mtROS, as the cell death inducers, are tightly regulated by mitochondria to maintain cell homeostasis [24]. While in breast cancer cells, [Ca2+]m overload and mtROS overproduction were achieved by RY10-4 treatment, which was able to further activate the mitochondrial apoptosis. As expected, we consistently observed the increase of apoptosis in RY10-4 treated breast cancer cells and tumors (shown in Figs. 3 and 8). Besides, the expression variations of apoptotic regulators in mitochondrial apoptosis were also explored by western blot, showing a consistent trend as described in previous studies [34]. To be detailed, the anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xl were displaced upon activation, permitting the pro-apoptotic Bax and Bcl-2 antagonist/killer 1 (Bak) to translocate to the outer mitochondrial membrane (OMM) and regulate OMM permeabilization. This was followed rapidly by the pore formation, and subsequent release of the mitochondria-resided apoptogenic factors (e.g. cytochrome c, AIF and Smac/DIABLO) into the cytosol. Subsequently, caspase-9 mediated apoptosis by assembling an apoptosome complex through the interaction of procaspase-9 with Apaf-1, dATP/ATP, and mitochondrial released cytochrome c, which subsequently recruited procaspase-3 to the complex and efficiently activated caspase-3, a common effector caspase, by the caspase-9-mediated proteolytic cleavage and finally resulted in cell apoptosis [35, 36].
Since ER-mitochondria transfer accounts largely for mitochondrial Ca2+ uptake and its alterations play a crucial role in cancer progression [37, 38], it is necessary to explore the upstream mechanism for ER Ca2+ release or the possible endoplasmic reticulum stress in our future research. Besides, the quantitative analysis of MCU and its regulators including mitochondrial calcium uptake 1 (MICU1), mitochondrial calcium uptake 2 (MICU2) and mitochondrial calcium uniporter regulator 1 (MCUR1) [39] were not involved in the present research considering the complexity and uncertainty therein, which will be taken into consideration in our next work.