In the present study, we modified the well-known BALB-CTA by adding an additional treatment on day 32 for 72 h in order to establish an in vitro tumor therapy model, the BALB-TTM. The effectiveness was proven successfully with 4 well-established chemotherapeutic agents and furthermore, for the very first time, a combined treatment with metformin was tested. The results are surprising as they show that metformin could partly mitigate the effects of the chemotherapeutic agents and a deregulated glucose metabolism seems to be involved in this process.
In vitro cell transformation assays mimic different phases of the in vivo multi-step carcinogenesis process. They are used by chemical, cosmetic and pharmaceutical industries for more than 6 decades to screen agents for carcinogenicity (36). We have shown previously that the BALB-CTA is also combinable with different molecular biologic and biochemical methods, thus allowing to screen for molecular mechanisms (27, 28). In this case, the malignant cell transformation is induced by treatment with the tumor initiator MCA following the tumor promotor TPA. Consequently, transformed cells lose their contact inhibition, start to grow over the monolayer of non-transformed BALB/c cells and pile up to characteristic, multilayered cell foci. For the BALB-TTM, an additional therapeutic treatment was performed on day 32 for 72 h on already existing cell foci. A reduction of the number of type-III foci could hence indicate a chemotherapeutic potential of the tested substance. Compared to rodent studies, this assay is less time consuming, needs a lower amount of resources and has no ethical implications. Moreover, molecular modes of action could be investigated easily, standardized and compared between non-transformed and malignant transformed cells.
The anticancer effects of metformin are widely described in vitro and in vivo (37) and now, were also confirmed in the BALB-CTA. Comparable to diabetic patients who show lower incidences for developing cancer when taking metformin for years (3, 38), chronical treatment with 1 mM metformin decreases number of type-III foci significantly and shows a tumor preventive effect. At this point, the BALB-CTA offers a strong tool for further mechanistic studies. Moreover, when metformin was added in the late phase of the BALB-CTA on already existing cell foci, a chemotherapeutic effect was observed with a significant decrease in number of type-III foci. Although plasma concentrations of metformin in diabetic patients are in the lower range of 10 to 40 µM (39), it was shown that metformin accumulates highly in tissues of mice, especially in the gastrointestinal tract where concentrations were up to 50 times higher compared to plasma (40).
Despite the anticancer effects of metformin, its application as an adjuvant in tumor therapy offers conflicting results. Therefore, we established an in vitro tumor therapy model in order to investigate interactions between metformin and several chemotherapeutic agents. First, the applicability of the new BALB-TTM was proven successfully with four chemotherapeutic agents from different classes. In this case, treatment for 72 h was sufficient to decrease the number of type-III foci significantly even in non-toxic concentrations for Dtx, MMC and 5FU. Such an effect was observed for Dox only in toxic concentrations. Second, the combined therapy with metformin was tested. An evidence for the cytoprotective role of metformin was given already via the Resazurin assay as metformin could mitigate the cytotoxic effects of Dtx and MMC. In the BALB-TTM, such an chemoresistance-inducing effect was shown for Dox and Dtx. In various in vitro and in vivo studies metformin was shown to decrease Dox-induced cardiotoxicity and is considered as a promising approach for patients treating with Dox (19). Moreover, metformin could not only reduce the therapeutic concentration of Dox and diminish cardiotoxic side effects, but also shows synergistic anti-tumor effects for prostate (41) and breast cancer (19, 42–46) in different cell and mouse models. However, in the present study metformin could not improve the anticancer effects of Dox in the BALB-TTM. To the contrary, the number of type-III foci increased slightly but not significantly. Consequently, the therapeutic effect seems to be highly dependent on the type of tumor. For metastatic castration-resistant prostate cancer, Dtx is the first-line chemotherapeutic agent. Since the treatment is associated with considerable toxic side effects, there is a need for chemosensitizing agents and it was shown that metformin is able to improve the prognosis (47). However, in vitro studies with different prostate cancer cell lines treated with metformin and Dtx demonstate controversial results (20, 48). A clinical study regarding the combined effect of Dtx with metformin in patients with castration-resistant prostate cancer showed that metformin did not act as an chemosensitizer and could not improve prostate cancer specific or overall survival (49). In our study, metformin even offers reverse results as the therapeutic, foci-reducing effect in the BALB-TTM is mitigated. Taken together, the potential role for metformin in prostate cancer therapy remains controversial and seems to be dependent on many individual factors. Thereby, the BALB-TTM offers a potent tool to elucidate the molecular interactions between Dtx and metformin.
In order to explain our observed effects of the combined therapy with MMC and metformin, we have focused on glucose metabolism. A deregulated energy metabolism in general is characteristic for several tumor cells and especially the glucose metabolism seems to be a promising target for cancer therapy (50). MMC is a DNA cross linker that requires reductive activation (bioreduction) to exert its chemotherapeutic effects (51). As mentioned elsewhere (52), an enhanced glycolytic rate results in higher NAD(P)H and thiol levels. Consequently, the induced intracellular reducing environment is able to facilitate the bioreduction of MMC. The effect of metformin on energy metabolism varies highly depending on the cell type and status of transformation. Therefore, we investigated the impact of metformin on glucose consumption and AMPK activation in non-transformed BALB/c fibroblasts first. In line with the observed effect in muscle cells (9) and podocytes (8), metformin increases the glucose consumption in the BALB/c cells dose-dependently. However, even when glucose concentration reaches a minimum of 0.5 g/l, the AMPK becomes not activated. Therefore, metformin seems to impair glucose metabolism in the utilized cell line without affecting the cellular energy sensor AMPK.
Due to the observed increase in glucose consumption, we have expected a synergistic effect of metformin and MMC in the BALB-TTM. Despite the higher glycolytic rate, metformin induced resistance to MMC in our studies. Indeed, a higher glucose consumption after metformin treatment is described only for healthy, peripheral tissue (8, 9). For cancer cells, a converse effect with lower glucose consumption after metformin treatment was shown that is further described as an inhibition of the Warburg effect (12–16). Therefore, we measured glucose consumption during the BALB-TTM in non-transformed monolayer cells and in the mixed population with cell foci of malignant transformed cells. As expected, we observed an increased consumption after metformin treatment in non-transformed cells but surprisingly, this was also the case in the mixed population. In fact, the MCA/TPA treated cells show even a higher glucose consumption compared to the non-transformed monolayer cells. At this point, a major limiting factor is the co-existence of non-transformed BALB/c monolayer cells and the malignant transformed, foci forming cells. Thus, we cannot precisely investigate the specific effect of metformin on the malignant transformed cells and have to consider, that the increase in glucose consumption is only due to the non-transformed monolayer cells. Possibly, metformin did not increase glycolysis in malignant transformed cells of the BALB-TTM and therefore did not enhance the therapeutic effect of MMC. In order to clarify the specific effects of metformin on malignant transformed cells in the BALB-TTM, investigations in isolated malignant transformed cells are strongly necessary.