Two intertwining molecular events determine the point from which the irreversible process of cell death begins. These include mitochondrial membrane permeability and caspase activation [
21], regardless of the apoptosis pathway [
10]. In the case of increase mitochondrial membrane permeability caused by e.g., overproduction of ROS (Reactive Oxygen Spieces) or overloading of organelles with Ca
2+ ions due to their increased uptake from the cytoplasm, it comes to the creation of megachannels in the mitochondrial membrane and lead to high permeability. The consequence of increasing mitochondrial membrane permeability is the release into cytoplasm of mitochondrial proapoptotic proteins (e.g., cytochrome c, SMAc/DIABLO, and AIF). Cytochrom c released from the membrane interstitial space of mitochondria interacts with the APAF-1 protein and changing its conformation leads to the formation of apoptosome complex that activates caspase-9, which in turn activates executive caspases-3, -6, -7 [
11]. Membrane permeability to cytochrome c is considered a critical step in the progression of irreversible apoptosis process, and we can conclude that this is an early stage of apoptosis [
22]. Here, we report that lycopene significantly affected the number of viable cells after 48 h incubation, and the percentage of living cells with depolarized mitochondrial membrane was considerably lower at 50 μM than in the control. This may suggest that the cells enter into early stage of apoptosis associated with a change in the structure of the mitochondrial membrane. The total percentage of apoptotic cells markedly increased in the 48-hour incubation with the 50 μM lycopene dose compared to 24-hour incubation. In our study, we did not show statistically significant differences after 24 h incubation with examined substances. Taking into consideration caspase/3/7 activity, we also do not observe statistically significant differences after 24 and 48-h lycopene incubation. Nevertheless, Palozza et al. found that a 24-hour treatment with lycopene resulted in a dose-dependent increase in 7-amido-4-methylcoumarin fluorescence, leading to activation of caspase-3 in prostatic carcinoma LNCaP cells [
23]. Puri et al. in their study conducted a randomized placebo trial, in which 50 patients with high-grade gliomas were treated surgically and then treated with oral lycopene 8 mg/day or placebo. Time to progression was longer in the lycopene-supplemented group but not statistically significant [
24]. Therapeutic benefits of using lycopene are well known in the case of various cancers. In the study conducted in 1995, authors demonstrated that the consumption of carotenoid was not associated with reduced risk of prostate cancer, but high lycopene intake was related to statistically significant risk reduction. Giovannuci et al. identified lycopene as a carotenoids with the greatest ability to inhibit the development of prostate cancer [
25]. In a similar study lasting from 1986 to 1998, 47,367 men were examined and completed a dietary questionnaire every four years. Frequent consumption of tomatoes was associated with a reduced risk of prostate cancer and the consumption of tomato sauce was associated with even lower risk of this type of cancer [
16]. Rafi et al. showed that lycopene in combination with a chemotherapeutic agents induced apoptosis in prostate cancer cells [
17]. In case of HT-29 colon carcinoma cells, it was shown that lycopene at concentration of 2 μM together with 25 μM eicosapentaenoic acid (EPA) synergistically inhibit tumor cell proliferation. Lycopene and EPA also blocker of Akt/mTOR activation contributing to decreased cell proliferation [
26]. In turn, in another study, the effects of lycopene on the proliferation of several human cell lines, including cancer, non-cancer cells were analyzed. The hepatic adenocarcinoma cells showed a decrease in proliferation rate in high doses of lycopene after 24 h and the non-neoplastic pulmonary cells showed a proliferation decreasing at the highest dose 10 mM after 72 hours, compared to the control. Cells of skin cancer, prostate cancer, breast cancer, lung cancer, and noncancerous skin did not show reduced proliferation [
27]. In the present study, there were no essential differences in the lycopene glioblastoma proliferation (data not shown), which is in accordance with a study performed by Burgess et al. [
27]. Another confirmation of the inhibitory effects of lycopene is the data presented by Karas et al., in which stimulation of IGF-1 (insulin-like growth factor-1) breast cancer cell growth has been significantly reduced by lycopene at 3 μM concentration. It was concluded that the inhibitory effect of lycopene on MCF7 cell growth is not due to carotenoid toxicity, but rather to interference in IGF-1 receptor signalling and cell cycle progression [
28]. Lycopene exerted a significant dose-related effect on the proliferation capacity of the myeloid leukemia, colon cancer, and Burkitt’s lymphoma cell line. This effect was observed in lymphocytic leukemia cells only at the highest dose (4 μM) used in that study. Increased apoptosis was found after incubation of colon cancer cells with 2 μM/mL and 4 μM/mL of lycopene as well as in Burkitt’s lymphoma cells after incubation with 2 μM. It has been concluded that the antiproliferative activity of lycopene on tumor cells and its effect on the rate of apoptosis depends on its dose and type of malignant cells [
29].
Gingerol, besides lycopene, exhibited potential anticancer activity in many types of cancer. Our results suggest that gingerol also has a significant influence on the depolarization of the mitochondrial membrane of tumor cells. At the highest concentration (500 μM), the percentage of dead cells after 24 h was 70%, and, after 48 h, it was 60%. The percentage of proliferating cells was lowest in culture with 50 μM [6]-gingerol; however, the values were comparable (data not shown). Similar results were obtained after a 48 h incubation. We presented, after 24-hour incubation, the highest percentages of late stage of apoptotic cells with caspase-3/7 activity or dead cells at the highest concentration of [6]-gingerol. We can suggest late apoptosis events even at the lowest concentration. Mostly, the dose-dependent activation of caspase-3/7 in this study confirmed apoptosis as the major mechanisms of cell death in U118MG treated with [6]-gingerol as evidenced by the increase of percentages of dead cells with a depolarized mitochondrial membrane. The result maintained was confirmed after 48-hour incubation. Moreover, after 24 and 48-h, we also observed a significant decrease in the percentage of viable cells observed with and increased [6]-gingerol concentration. Research conducted by Lee et al. provided that gingerol positively influences apoptosis GBM cell lines U87, U343, and T98G through TRAIL (TNF-related apoptosis-inducing ligand) [
30]. The potential of [6]-gingerol and its synergistic treatment with therapeutic agents has been evaluated for cervical adenocarcinoma, leading to the inhibition growth of HeLa cells, induced cell cycle arrest in the G0/G1 phase, and induced apoptosis [
31]. However, in the study on the adenocarcinoma A549 cell line, [6]-gingerol had no effect on cell proliferation [
32]; similar results were obtained in our research. Studies carried out on MCF-7 and MDA-MB-231 cell lines after incubation with ginger extract and on prostate cancer cell line PC3R after stimulation with [6]-gingerol with shogalou at 100 μM/mL concentration clearly indicate inhibited cell proliferation [
33,
34]. The antiproliferative and proapoptotic properties of gingerol on retinal tumor cells were confirmed by Meng et al.; in his study on RB355 cell line, it was found that this antineoplastic effect is due to the ability to induce apoptosis, cell cycle inhibition, and Pi3K/Akt signal regulation [
35]. The studies conducted by Lee at al. confirmed anticancer and proapoptotic properties of gingerol; the results indicate that [6]-gingerol has the ability to inhibit cyclin D1, which is protooncogene found in many cancers. This compound induced the expression of NAG-1 proapoptotic cytokine in the colon carcinoma cell line [
14]. This also confirms the conclusions of our own research on pro-apoptotic properties of [6]-gingerol. Referring to anticancer effects of silymarin, it was noticed that silymarin inhibited oral tumor growth and proliferation of tumor cells [
15,
36].
Ranjbar et al. showed that treatment of Ramos cancer cells with 100 μg/mL of sylimarin markedly increased the activity of caspase-3 [
37]. Montgomery et al. showed that silymarin at the highest dose of 100 μM/mL has antiproliferative activity in colorectal cancer cell lines: DLD-1, LoVo, and HCT116 [
38]. Furthermore, the experiment carried out on the ovarian cell line indicates the inhibitory properties of silymarin: significantly limits the growth and rate of proliferation, inhibits the cell cycle in G1/S phase, and the doses of 50 μg/mL and 100 μg/mL activate the apoptotic pathway and induce cytochrome C release, as well as the reduction of the mitochondrial membrane potential [
39]. It was found that 20 μM and 50 μM concentrations almost completely blocked tumor cell invasion, inhibiting cell proliferation and migration of glioblastoma cells. On the other hand, silymarin stimulates the process of apoptosis by activating caspases [
40]. It is also a confirmation of the results of our own study, where it was observed that self-acting silymarin induces the death of glioblastoma cells.