3.1. Acidic Polyphenols from R. canina Enhance the Antiproliferative Effect of Gold Complex
The antiproliferative effect of the acidic polyphenols fraction (AP) of rosehips from R. canina
on Caco-2 cell line after 72 h of incubation were previously evaluated by us [17
], reading an IC50
value of 125 μg/mL. On the other hand, the effect of the gold complex (Au(C≡C-2-NC5
)(PTA)) towards the same cell model displayed a higher in vitro effect than plant extracts with an IC50
value of 3.8 μM [16
Initially, a range of concentrations of acidic polyphenols (125, 62.5 and 31.25 μg/mL) was tested in combination with the gold complex at its IC50
value for 72 h (Figure 1
A). Results obtained showed the key role of AP concentration on the antiproliferative effect of drug combination, since all the tested concentration values decreased cell viability in comparison to both negative control and cells treated with the gold complex as a single agent (p
< 0.05). In order to analyze whether the observed results were a consequence of synergy or additivity, the kind of pharmacological interaction between single agents was further studied with an isobologram (Figure 1
B), after re-calculation of the corresponding IC50
values of the gold complex in the presence of both concentrations of AP (62.5 and 31.25 μg/mL), which afforded the IC50
values of 1.25 and 2.04 μM respectively, versus the previous value of 3.8 μM obtained with the gold complex alone. As can be observed on Figure 1
B, the combination of the gold complex and AP showed additive cytotoxicity on Caco-2 cell line.
Then, time-course analyses were performed after 24, 48 and 72 h of incubation (Figure 1
C). Incubation time was found to be crucial for the antiproliferative effect of the drug combination, since it induced significant changes in cell viability (p
< 0.05) at short-time incubation (24 h) whereas for the gold complex these changes were not observed until 48 h. Moreover, the drug combination induced a 50% reduction on cell viability upon 48 h of incubation, whereas single agents reached that effect after 72 h.
Caco-2 cells undergo spontaneous differentiation after reaching confluence and acquire the morphology and enzymatic profile of a mature enterocyte [22
]. Thus, differentiated Caco-2 cells are considered as an acceptable model of the intestinal barrier and have been traditionally used to evaluate the toxicity of novel drugs on a non-cancerous model [23
]. The drug combination and AP did not induce significant changes on cell proliferation of differentiated Caco-2 cells after long-term incubation (72 h) (Figure S2
), which might suggest that such combination displays selectivity for this cancer type. It has been reported that plant extracts and plant metabolites might display a different behavior on cancer and non-cancer cells. As a part of normal diet, they are well-tolerated by non-cancer cells, whereas are harmful for cancer cells. Ivanova et al. [24
] observed resveratrol-induced cell death of Jurkat cells but not influenced cell viability rates of normal lymphocytes. In this context, plant extracts have been proposed as co-adjuvants to ameliorate side effects caused by chemotherapeutic drugs such as cisplatin given their antioxidant and anti-inflammatory properties [25
]. In the specific case of differentiated Caco-2 cells, Di Nunzio et al. [27
] found that olive pomace successfully protected this intestinal barrier model from inflammation induced by the addition of interleukin-1β. Therefore, it is feasible to suppose that R. canina
extract might protect differentiated Caco-2 cells by the oxidative and/or inflammatory damage induced by the gold complex.
3.2. Drug Combination Triggers Apoptosis on Caco-2 Cells
We found it interesting to analyze the type of cell death induced by the combination of the extracts and the gold complex on undifferentiated Caco-2 cells.
After 48-h incubation with drug combination, 2.8-fold increase in late apoptotic population and a 2.5-fold increase in early apoptotic cells were observed, whereas no significant changes in the necrotic cell population were found (Figure 2
A). Furthermore, a 5.44-fold increase in active caspase 3 (Figure 2
B) along with cell cycle arrest on G1
phase (Figure 2
C) were observed at 48 h. Taken together, these results suggest that the drug combination induced apoptosis on Caco-2 cells. Its pro-apoptotic effect might be a direct consequence of the concomitant administration of the gold complex and the acidic polyphenols fraction, since incubation with single agents did not induce significant changes on apoptotic cells population after 48 h of incubation (Figure S3
In a previous work [16
], we reported that 24 h incubation with the gold complex (Au(C≡C-2-NC5
)(PTA)) induced Caco-2 cell death by a programmed form of necrosis called necroptosis, which is caspase-independent. Interestingly, treatment of cell culture with the drug combination for 48 h did not modify the number of cells with activated receptor–interaction protein kinase 1 (RIP-1), one of the key regulators of necroptosis [28
], which suggests that the necroptotic pathway is non-activated by the drug combination (Figure S4
). Moreover, pre-incubation of cell culture with the RIP-1 inhibitor necrostatin-1 (Nec-1) (50 μM, 1h) resulted in no significant changes in cell viability in comparison with the administration of drug combination in the absence of Nec-1 (Table 1
), which further confirmed that no necroptosis was triggered by the drug combination.
Shen and Codogno [29
] found that the inhibition of necroptosis led to cell death by apoptosis. In light of this finding, the high rates of apoptotic cells observed on Figure 2
A might be a consequence of the disruption of the necroptotic process induced by the concomitant administration of acidic polyphenols and the gold complex. In line with this, autophagy seems to play a key role in both inhibition and promotion of other forms of cell death given its dual cytoprotective and cytotoxic nature. Some authors have reported that activation of cytoprotective autophagy might be related to necroptosis inhibition [30
]. Since 48 h of treatment with the drug combination resulted in no evidence of necroptosis activation, a likely activation of cytoprotective autophagy must be an upstream event. Therefore, changes in autophagosome formation were analyzed after 24 h incubation. As can be observed in Figure 3
A, the drug combination induced a 1.5-fold increase in autophagosomes formation, whereas the gold complex and AP separately did not induce any significant changes (Figure S5
). Consequently, the concomitant administration of both drugs triggers autophagy, which might be related to the absence of necroptosis previously discussed.
Interestingly, pre-incubation of Caco-2 cells with the autophagy inhibitor chloroquine (CQ) (10 μM, 1 h) resulted in no significant changes in cell viability when comparing with cells non-treated with CQ after 24 h or 72 h treatment with the drug combination (Figure 3
B). The autophagic process consists of two stages: the early stage comprises the formation of the autophagosome, which engulfs damaged subcellular structures as well as protein aggregates, whereas in the late stage the fusion of autophagosome and lysosome ends with the digestion of the non-functional cellular components [32
]. Given that CQ inhibits late autophagy by preventing the autophagosome–lysosome fusion, our data suggest that the autophagic process is initiated upon treatment with the drug combination, although autophagy might not display a significant role on its antiproliferative effect. In fact, uncompleted autophagy has been related to high rates of apoptosis due to the accumulation of non-functional cellular structures [33
]. Therefore, it is feasible to hypothesize that the observed strong pro-apoptotic effect induced by the drug combination (Figure 2
A) might be a consequence of a blockage of the autophagic flux.
3.3. Drug Combination Disrupts Redox Homeostasis, Which Induces Mitochondrial Disturbances along with Lysosomal Dysfunction
In an effort to determine whether treatment with the drug combination triggered uncompleted autophagy, we analyzed lysosomal integrity 24 h and 48 h after incubation (Table 2
). We observed a time-dependent loss of acidification that correlates with lysosomal impairment. As aforementioned, drug-mediated induction of lysosome alkalization has been related to a blockage of the autophagic flux and high rates of apoptosis [33
], and mitochondrial dysfunction is considered to be one of the main causes of the impairment of lysosomes [35
]. With this in mind, changes in mitochondrial membrane potential (ψm
) were analyzed after 24 h and 48 h incubation with the drug combination (Table 2
). We found a time-dependent increase in cell population with an altered value of ψm
, which suggests a disruption on normal mitochondrial function. Since a significant loss of ψm
was noticed after 24 h, prior to lysosomal alkalization (Table 2
), mitochondrial dysfunction seems to be an upstream event relative to lysosomal impairment.
Finally, we measured reactive oxygen species (ROS) levels due to their key role in mitochondrial homeostasis. A significant increase in ROS production was observed after a short incubation time (1 h) and it was maintained upon overnight treatment with the drug combination (Table 3
). The role of ROS on cell death was further confirmed by pre-incubation of Caco-2 cells with the ROS scavenger N-acetylcysteine (NAC) (3 mM, 1 h), which reduced the great decrease in cell viability caused by 72 h treatment with the drug combination (Table 3
). Therefore, our data suggest that disruption of redox balance and mitochondrial dysfunction with lysosomal impairment and autophagy blockage triggered by drug combination, results in apoptotic cell death.