Calcium Regulates HCC Proliferation as well as EGFR Recycling/Degradation and Could Be a New Therapeutic Target in HCC

Calcium is the most abundant element in the human body. Its role is essential in physiological and biochemical processes such as signal transduction from outside to inside the cell between the cells of an organ, as well as the release of neurotransmitters from neurons, muscle contraction, fertilization, bone building, and blood clotting. As a result, intra- and extracellular calcium levels are tightly regulated by the body. The liver is the most specialized organ of the body, as its functions, carried out by hepatocytes, are strongly governed by calcium ions. In this work, we analyze the role of calcium in human hepatoma (HCC) cell lines harboring a wild type form of the Epidermal Growth Factor Receptor (EGFR), particularly its role in proliferation and in EGFR downmodulation. Our results highlight that calcium is involved in the proliferative capability of HCC cells, as its subtraction is responsible for EGFR degradation by proteasome machinery and, as a consequence, for EGFR intracellular signaling downregulation. However, calcium-regulated EGFR signaling is cell line-dependent. In cells responding weakly to the epidermal growth factor (EGF), calcium seems to have an opposite effect on EGFR internalization/degradation mechanisms. These results suggest that besides EGFR, calcium could be a new therapeutic target in HCC.


Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and the third most common cause of cancer death worldwide [1,2]. It is rarely detected early and is often fatal within a few months of diagnosis. Among the risk factors for the disease are hepatitis C and B virus infection, alcoholic liver disease, liver cirrhosis, tobacco smoking, and obesity [3]. Until now, surgery has played a central role in the treatment of liver cancer. However, HCC recurrence after surgical treatment is frequent and the long-term prognosis of patients with HCC is generally poor.
The disease has a complex molecular pathogenesis in which several signaling pathways could be involved [4][5][6]. In particular, various studies have demonstrated that growth factors, acting in part through intracellular calcium ([Ca 2+ ]i) release [7] play a pivotal role, and their signaling pathways are  To better understand the IC50 effect of Gefitinib (GEF) and AZD9291 (AZ) EGFR inhibitors (listed in Table 1) in signaling, starved cells were treated for 3 h with GEF IC50 or AZ IC50 and DMSO as control. GEF or AZ treatment switched off EGFR, ERK, and AKT phosphorylations in all cell lines analyzed. EGF was not able to rescue AKT and ERK phosphorylation following GEF or AZ EGFR inhibition ( Figure 2; Figure S2). To better understand the IC50 effect of Gefitinib (GEF) and AZD9291 (AZ) EGFR inhibitors (listed in Table 1) in signaling, starved cells were treated for 3 h with GEF IC50 or AZ IC50 and DMSO as control. GEF or AZ treatment switched off EGFR, ERK, and AKT phosphorylations in all cell lines analyzed. EGF was not able to rescue AKT and ERK phosphorylation following GEF or AZ EGFR inhibition (Figure 2; Figure S2).  Figure 1A; numbers in the abscissa refer to the corresponding lane in panel A. p value < 0.05 (*); p value < 0.01 (**); p value < 0.001 (***).
Moreover, as observed in Figure 3, both drugs (GEF and AZ) blocked the activated EGFR signaling, after as little as 30 min of incubation, in all the cell lines analyzed, even though the EGFR phosphorylated form was still present. Unlike in the other cells, in HUH-6 cells both GEF and AZ had already reduced the EGFR phosphorylation within 30 min, but the downstream pathways were only weakly affected as compared to the other cell lines.
Moreover, as observed in Figure 3, both drugs (GEF and AZ) blocked the activated EGFR signaling, after as little as 30 min of incubation, in all the cell lines analyzed, even though the EGFR phosphorylated form was still present. Unlike in the other cells, in HUH-6 cells both GEF and AZ had already reduced the EGFR phosphorylation within 30 min, but the downstream pathways were only weakly affected as compared to the other cell lines. stabilization of the receptor in its inactive form [60,64,65], once again except for the HUH-6 cell line. It may be speculated that the internalization/degradation mechanism in the HUH-6 cell line is different from that observed in the other cell lines (Figure 3; Figure S3 A and B).
In HepG2 and HUH-6 cell lines EGFR trafficking seems to be less evident than in HUH-7 and Hep3B cells. Moreover, it is noteworthy that treatment with GEF in Hep3B cells leads to a rescue of pAKT within 3 to 6 h, as seen in Figure 3 (see also Figure 2).  Table 1) (DMSO as control). Treatments were performed for 30 min, 3 h and 6 h.
As expected, following EGFR signaling switch-off by GEF or AZ, the proliferation of all the cell lines analyzed was negatively affected by both drugs (Figure 4).
In conclusion, both AZ and GEF act on the same pathways downstream of EGFR (pAKT and pERK) and both of them are able to switch off already activated EGFR pathways, as may be expected in the in vivo context. However, AZ activity was stronger than GEF activity even at lower concentrations, both in signaling and in proliferation assays, as indicated also by their IC50 values in Table 1 Figure 3. Western blot panels of HepG2, HUH-7, HUH-6, and Hep3B starved cell lines stimulated with 100 ng/mL of EGF for 30 min before and during treatment with GEF or AZ IC50 (as indicated in Table 1) (DMSO as control). Treatments were performed for 30 min, 3 h and 6 h.
As described in literature, 3 or 6 h later, EGF-stimulated cells (DMSO lane in Figure 3) undergo EGFR internalization and lysosomal degradation (a phenomenon called "EGF-dependent EGFR degradation", as indicated by the total EGFR (EGFR TOT) level reduction) [57][58][59][60][61][62][63]. The same was not observed in the HUH-6 cell line that showed an even more robust EGFR phosphorylation until 6 h, followed by no reduction of EGFR levels.
At 3 and 6 h, EGF-stimulated cells treated with EGFR inhibitors showed a reduced internalization/degradation of the receptor compared to untreated cells, with a consequent stabilization of the receptor in its inactive form [60,64,65], once again except for the HUH-6 cell line. It may be speculated that the internalization/degradation mechanism in the HUH-6 cell line is different from that observed in the other cell lines ( Figure 3; Figure S3A,B).
In HepG2 and HUH-6 cell lines EGFR trafficking seems to be less evident than in HUH-7 and Hep3B cells. Moreover, it is noteworthy that treatment with GEF in Hep3B cells leads to a rescue of pAKT within 3 to 6 h, as seen in Figure 3 (see also Figure 2).
As expected, following EGFR signaling switch-off by GEF or AZ, the proliferation of all the cell lines analyzed was negatively affected by both drugs (Figure 4).
In conclusion, both AZ and GEF act on the same pathways downstream of EGFR (pAKT and pERK) and both of them are able to switch off already activated EGFR pathways, as may be expected in the in vivo context. However, AZ activity was stronger than GEF activity even at lower concentrations, both in signaling and in proliferation assays, as indicated also by their IC50 values in Table 1.  Table 1), DMSO as control. Data are plotted in the graph as normalized by DMSO.

EGFR Signaling and Calcium Chelators
Preliminary proliferation assays carried out on these HCC cell lines in the presence of EGF added to the serum-free medium showed an unexpected regulation of cell growth. Results suggested two possibilities: besides EGF, the component responsible could be produced by cells, and released into the culture medium (with a paracrine and antiproliferative activity), or might be already present in the culture medium and consumed over time (with a pro-proliferative activity). One of these last factors was calcium. Therefore, it was hypothesized that calcium ions could be actively involved in regulating EGFR-dependent HCC cells growth.
In order to investigate this hypothesis, HUH-7 and HUH-6 cell line proliferation was measured in starved cells (0% FBS), treated or not with EGF, in the presence of 2 mM EDTA solution as calcium chelator ( Figure 5). 24 h of EDTA treatment negatively affected cell number in both cell lines within 72 h. On the contrary, 0.5% DMSO tended to increase the number of cells, mostly when added with EGF.
As widely acknowledged in literature, DMSO can induce transient water pores in cell membranes, increasing permeability, thus Ca 2+ can easily flow through these pores from the medium to the cytosol [66][67][68][69].  Table 1), DMSO as control. Data are plotted in the graph as normalized by DMSO.

EGFR Signaling and Calcium Chelators
Preliminary proliferation assays carried out on these HCC cell lines in the presence of EGF added to the serum-free medium showed an unexpected regulation of cell growth. Results suggested two possibilities: besides EGF, the component responsible could be produced by cells, and released into the culture medium (with a paracrine and antiproliferative activity), or might be already present in the culture medium and consumed over time (with a pro-proliferative activity). One of these last factors was calcium. Therefore, it was hypothesized that calcium ions could be actively involved in regulating EGFR-dependent HCC cells growth.
In order to investigate this hypothesis, HUH-7 and HUH-6 cell line proliferation was measured in starved cells (0% FBS), treated or not with EGF, in the presence of 2 mM EDTA solution as calcium chelator ( Figure 5). 24 h of EDTA treatment negatively affected cell number in both cell lines within 72 h. On the contrary, 0.5% DMSO tended to increase the number of cells, mostly when added with EGF.
As widely acknowledged in literature, DMSO can induce transient water pores in cell membranes, increasing permeability, thus Ca 2+ can easily flow through these pores from the medium to the cytosol [66][67][68][69]. The EDTA effect was observed also at molecular level by western blot on HUH-7 cells treated or not with 2 mM EDTA for 6 and 24 h ( Figure 6; Figure S4). Proliferative inhibition was confirmed also by a Cyclin D1 reduction, especially within 24 h of EDTA treatment. Following calcium subtraction EGF addition did not rescue pERK nor Cyclin D1 levels as early as 6 h, even though the pEGFR level was still high, suggesting that calcium is necessary for EGFR signaling propagation. Notably, within 6 h EDTA was able to induce a sustained EGFR downmodulation as compared to EGF alone. After 24 h, EGF-dependent EGFR degradation was almost complete even without EDTA.
The effect of EDTA on pAKT 24 h later was impressive. AKT phosphorylation dramatically increased, probably to counteract the EDTA-triggered apoptotic stimulus ( Figure 6A). DMSO was also used as positive control. As expected, 24 h of 0.5% DMSO treatment upregulated pERK and increased the Cyclin D1 levels more than EGF alone, indicating that intracellular free Ca 2+ acts through the ERK pathway ( Figure 6B).  The EDTA effect was observed also at molecular level by western blot on HUH-7 cells treated or not with 2 mM EDTA for 6 and 24 h ( Figure 6; Figure S4). Proliferative inhibition was confirmed also by a Cyclin D1 reduction, especially within 24 h of EDTA treatment. Following calcium subtraction EGF addition did not rescue pERK nor Cyclin D1 levels as early as 6 h, even though the pEGFR level was still high, suggesting that calcium is necessary for EGFR signaling propagation. Notably, within 6 h EDTA was able to induce a sustained EGFR downmodulation as compared to EGF alone. After 24 h, EGF-dependent EGFR degradation was almost complete even without EDTA.
The effect of EDTA on pAKT 24 h later was impressive. AKT phosphorylation dramatically increased, probably to counteract the EDTA-triggered apoptotic stimulus ( Figure 6A). DMSO was also used as positive control. As expected, 24 h of 0.5% DMSO treatment upregulated pERK and increased the Cyclin D1 levels more than EGF alone, indicating that intracellular free Ca 2+ acts through the ERK pathway ( Figure 6B). These results indicated that calcium ions are involved in the proliferative capability of HCC cell lines, as well as in EGFR degradation (calcium subtraction induced EGFR degradation within 6 h in an activated system).
To rule out the possible involvement of apoptotic signals triggered by EDTA, we replaced EDTA with the less toxic EGTA and examined AKT phosphorylation (pAKT) levels at a later time (24 h). Proteins extracted from cells treated with EDTA were loaded as positive control. Molecular analysis on pAKT levels excluded any apoptotic effect after 24 h of EGTA treatment ( Figure 7C; Figure S5). Moreover, also in this case the results obtained confirmed the calcium involvement. HUH-7 cells fate resulted dependent on calcium depending on their starting proliferative status. More in detail, in actively proliferating cells (10% FBS (48 h)) EGTA treatment reduced proliferation ( Figure 7A), while CaCl2 addition promoted cell proliferation and therefore cell cycle progression. On the contrary, in non-proliferating cells (serum-free (SF) medium (48 h)), calcium addition halved the number of viable cells, and as a consequence, EGTA in the presence of calcium rescued the number of viable cells ( Figure 7B).   These results indicated that calcium ions are involved in the proliferative capability of HCC cell lines, as well as in EGFR degradation (calcium subtraction induced EGFR degradation within 6 h in an activated system).
To rule out the possible involvement of apoptotic signals triggered by EDTA, we replaced EDTA with the less toxic EGTA and examined AKT phosphorylation (pAKT) levels at a later time (24 h). Proteins extracted from cells treated with EDTA were loaded as positive control. Molecular analysis on pAKT levels excluded any apoptotic effect after 24 h of EGTA treatment ( Figure 7C; Figure S5). Moreover, also in this case the results obtained confirmed the calcium involvement. HUH-7 cells fate resulted dependent on calcium depending on their starting proliferative status. More in detail, in actively proliferating cells (10% FBS (48 h)) EGTA treatment reduced proliferation ( Figure 7A), while CaCl 2 addition promoted cell proliferation and therefore cell cycle progression. On the contrary, in non-proliferating cells (serum-free (SF) medium (48 h)), calcium addition halved the number of viable cells, and as a consequence, EGTA in the presence of calcium rescued the number of viable cells ( Figure 7B).  On the basis of these results, we tested EGTA treatment in association with the two EGFR inhibitors, GEF and AZ, at their respective IC50 values. In proliferating cells (whose proliferative capability is shown by the increased number of viable cells in the CTR medium bar versus the T0 bar), the combined treatment reduced the proliferative capability of cells using either GEF and AZ ( Figure 8A). Conversely, as expected, in non-proliferating cells (the CTR medium bar and T0 bar displayed no differences) EGTA combined with either drug led cells to maintain or even slightly increase their proliferative capacity, reverting the trend observed in proliferating cells ( Figure 8B). However, both calcium chelators, EDTA and EGTA, do not only act on calcium ions outside the cell, but are also able to form complexes with different cations. For these reasons, we focused on the cell-permeable and specific intracellular Ca 2+ chelator BAPTA-AM in order to manipulate the intracellular Ca 2+ free levels.
In the HUH-7 cell line, 6 h of 10 µM BAPTA_AM treatment reduced the EGF-induced Cyclin D1 increase, even though the EGFR phosphorylation was still sufficient.
Moreover, in BAPTA_AM treated cells, after 6 h pAKT was higher than in EDTA treated cells, but within 24 h, in BAPTA_AM treated cells, it returned to the basal level and was strongly increased in EDTA samples, indicating that BAPTA_AM does not trigger the apoptotic pathway ( Figure 9A,B; Figure S6A).
It is noteworthy that within 6 h, the combined treatment, BAPTA_AM and EGF, was able to induce a greater EGFR level reduction than EGF stimulation alone, emphasizing the important role of calcium ions in EGFR recycling. Thus, calcium subtraction triggered EGFR lysosomal degradation ( Figure 9A,B; Figure S6A).
On the contrary, HUH-6 cells treated with BAPTA_AM and EGF behave differently from what was observed in HUH-7 cells. In line with what is shown in Figure 3, BAPTA_AM and EGF treatment increased the levels of EGFR within 6 h, suggesting that calcium is necessary for degradation (instead of recycling) of the EGFR activated form ( Figure 9C; Figure S6B).  However, both calcium chelators, EDTA and EGTA, do not only act on calcium ions outside the cell, but are also able to form complexes with different cations. For these reasons, we focused on the cell-permeable and specific intracellular Ca 2+ chelator BAPTA-AM in order to manipulate the intracellular Ca 2+ free levels.
In the HUH-7 cell line, 6 h of 10 µM BAPTA_AM treatment reduced the EGF-induced Cyclin D1 increase, even though the EGFR phosphorylation was still sufficient.
Moreover, in BAPTA_AM treated cells, after 6 h pAKT was higher than in EDTA treated cells, but within 24 h, in BAPTA_AM treated cells, it returned to the basal level and was strongly increased in EDTA samples, indicating that BAPTA_AM does not trigger the apoptotic pathway ( Figure 9A,B; Figure S6A).
It is noteworthy that within 6 h, the combined treatment, BAPTA_AM and EGF, was able to induce a greater EGFR level reduction than EGF stimulation alone, emphasizing the important role of calcium ions in EGFR recycling. Thus, calcium subtraction triggered EGFR lysosomal degradation ( Figure 9A,B; Figure S6A).
On the contrary, HUH-6 cells treated with BAPTA_AM and EGF behave differently from what was observed in HUH-7 cells. In line with what is shown in Figure 3, BAPTA_AM and EGF treatment increased the levels of EGFR within 6 h, suggesting that calcium is necessary for degradation (instead of recycling) of the EGFR activated form ( Figure 9C; Figure S6B). In order to confirm the EGFR degradation through proteasome machinery, the proteasome machinery inhibitor MG132 was used. The HUH-7 and HepG2 cell lines reduced the EGFR levels 6 h of EGF stimulation, as seen above. BAPTA_AM further emphasized this result whereas MG132 rescued the EGFR levels. These results indicate that in both cell lines the EGF-induced EGFR degradation is reinforced by calcium subtraction after as little as 6 h, and it is triggered by proteasome.
Once again, HUH-6 cells showed the opposite effect. In EGF-stimulated cells, BAPTA_AM increased the EGFR levels, as if the reduction of calcium level blocked degradation in favor of recycling and MG132 inverted the effect, reducing the levels of EGFR (Figure 10; Figure S7 A   In order to confirm the EGFR degradation through proteasome machinery, the proteasome machinery inhibitor MG132 was used. The HUH-7 and HepG2 cell lines reduced the EGFR levels 6 h of EGF stimulation, as seen above. BAPTA_AM further emphasized this result whereas MG132 rescued the EGFR levels. These results indicate that in both cell lines the EGF-induced EGFR degradation is reinforced by calcium subtraction after as little as 6 h, and it is triggered by proteasome. Once again, HUH-6 cells showed the opposite effect. In EGF-stimulated cells, BAPTA_AM increased the EGFR levels, as if the reduction of calcium level blocked degradation in favor of recycling and MG132 inverted the effect, reducing the levels of EGFR ( Figure 10; Figure S7A,B).

Discussion
The main aim of this study was to find a second target involved in the EGFR pathway to be targeted alone or in combination with current therapies (TKIs) in order to reduce TKIs dosage and hence their side effects, as well as the multi-drug resistance often acquired by cancer cells.
We first tested the AZD9291 on the HCC cell lines studied, showing that its activity was stronger than that of Gefitinib, even at lower doses. AZD9291 has been developed as a third-generation irreversible inhibitor with selectivity against T790M mutant versus wild type EGFR. However, on the HCC cell lines analyzed, the inhibition of proliferation was greater with AZD9291 than Gefitinib, even though all the cell lines tested harbored the wild type form of EGFR. As already demonstrated for Gefitinib, AZD9291 also acts on AKT and ERK pathways.
On the basis of our previous observations, we noticed that EGF-dependent HCC proliferation was governed also by a second factor. After having excluded the presence of some apoptosis-induced factors produced and released by cells, we postulated that calcium ions could be involved in this process. To investigate this possibility, we used different calcium chelators. First results observed using EDTA showed a great involvement of calcium both in EGFR-dependent proliferation and in EGFR signaling. To corroborate these results we treated cells with DMSO. As already described in literature, DMSO can induce water pores in dipalmitoyl-phosphatidylcholine bilayers through which ions can penetrate inside the cell [70]. Through these pores, it becomes easier for calcium ions to pass in and out of the cell and thus to function as positive regulators of cell proliferation [71,72]. In line with this, DMSO treatment enhanced proliferation, activating the pro-proliferative pathways, especially when added together with EGF.
However, EDTA upregulated the AKT phosphorylation independently of EGF, demonstrating its high apoptotic potential [73,74]. For this reason, we moved on to the EGTA calcium chelator. Unlike EDTA, EGTA showed no toxicity. It was noteworthy that within only 6 h of combined EGTA plus EGF treatment, the levels of EGFR were markedly reduced, more than by EGF alone. After 24 h of EGF treatment alone the EGFR downmodulation was almost complete. This stimulus-induced trafficking is already known as EGF-dependent EGFR internalization and is aimed at regulating the timing of EGFR signaling. In this work, by extracellular calcium withdrawal, we were able to anticipate the phenomenon from 24 to 6 h. Our results were confirmed using the more specific cytoplasmic calcium ions chelator BAPTA_AM.
Taken together, these results show that intracellular free calcium, necessary for EGFR signal propagation to occur inside the cell, is also a key regulator of cell cycle progression or apoptosis in HCC cell lines, depending on their proliferative status at that specific time point. More in detail, calcium enhances proliferation in already proliferating HCC cells whereas it pushes non-proliferating (G0) HCC cells towards apoptosis.
Besides its role in the cell cycle, we observed for the first time that after as little as 6 h treatment, calcium is essential also for the active EGFR fate: recycling or degradation. To distinguish between the two EGFR events, proteasome inhibition by MG132 was necessary. More in detail, in EGF-sensitive cells, such as HUH-7, HepG2, and Hep3B, intracellular calcium subtraction facilitates EGF-induced EGFR degradation, that is otherwise visible only much later (24 h), indicating that in these cells recycling is calcium-dependent. Conversely, HUH-6 cells did not only seem to be less sensitive to EGF stimulus but also showed an increased EGFR-recycling following BAPTA_AM treatment, suggesting that in this cell line calcium positively regulates EGF proteasomal degradation rather than recycling.
However, in both cases combined therapy with Gefitinib or AZD9291 and BAPTA_AM reduced the HCC cell viability, also with half doses of TKIs. For the purposes of avoiding MDR acquisition by HCC, this result is really significant if we take into consideration the fact that, as demonstrated, a sustained increase of intracellular calcium is actively related to MDR in HCC and the inhibition of calcium enhanced the efficacy of chemotherapy [75]. Moreover, early EGFR internalization and its consequent signaling down-modulation could be of great relevance in HCC treatment. Indeed, as shown in Figure 8A,B), in G0 phase cells calcium subtraction tended to reduce the drug activity. On the contrary, in actively proliferating cells, TKIs increased their antiproliferative ability following calcium chelation. Considering that the in vivo tumoral condition is closer to an actively proliferating than to a quiescent system, it may be that TKIs could be more effective if administered together with an intracellular calcium chelator. However, calcium chelator administration in vivo in xenograft HCC models requires further accurate, close investigation in order to guarantee a correct delivery of the drug and avoid its release in body districts strongly dependent on calcium for their function (such as the muscle or cardiac systems).

Protein Extraction
Cellular protein was extracted using RIPA lysis and extraction buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease and phosphatase inhibitors. Cell pellets were lysed for 30 min, and samples were centrifuged at 12,000 rpm for 20 min at 4 • C in a microcentrifuge. The supernatant liquid was collected in new Eppendorf tubes and stored at −20 • C.
Protein concentration was measured using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Cell Viability Assay
Cell viability was measured using the Sulforhodamine B (SRB) assay, which is based on the stoichiometric binding of SRB dye to proteins under mild acidic conditions and its subsequent extraction under basic conditions. The amount of dye extracted is a proxy for cell mass and thus the number of cells in a sample. The absorbance of the dye in solution is measured at OD 565 nm using an automated microplate reader (Perkin Elmer, Waltham, MA, USA). Sulforhodamine B sodium salt (S9012) was purchased from Sigma.

Western Blot Analysis
40 µg proteins per sample were separated using 4-20% SDS-PAGE and transferred to 0.2 µm nitrocellulose Trans-blot turbo TM membranes (Bio-Rad Laboratories Inc.) using the Bio-Rad electrotransfer system (Bio-Rad Laboratories Inc.). The membranes were blocked with blocking buffer (mixed 5% non-fat dry milk, 150 mM NaCl, 0.1% Tween-20 and 20 mM Tris-HCl and adjusted to a pH of 7.6) for 1 h at room temperature and probed with specific primary antibodies at 4 • C overnight. The protein bands were detected with HRP-conjugated secondary antibodies for 1 h at room temperature using custom-made ECL TM Prime Western Blotting Detection Reagents (Amersham GE, Little Chalfont, UK). The Image Lab TM software digital imaging system ChemiDoc TM XRS+ (Bio-Rad Laboratories Inc.) was used to detect the target protein on immunoblot nitrocellulose membranes.

Statistical Analysis
Plotted values are shown as means ± standard deviation. Statistical significance of the results was determined using the two-tailed unpaired Student's t test to determine whether the two datasets were significantly different. A value of p < 0.05 was considered significant.
For cell survival data of Figure 4, data from GEF and AZ treatments were compared, fitting a linear model for each subgroup of samples. The differences between estimated coefficients were assessed through the generation of a model which also contemplates an interaction term between the variable days and drug. The p-value obtained provides confidence about the generalizability of the differences in linear trends observed for GEF and AZ treatments.

Conclusions
Considering the plethora of calcium activities in the liver, as well as the fact that calcium channels are overexpressed in many HCC, where calcium plays a role in inducing MEK/ERK-triggered proliferation, calcium manipulation in HCC cells may have a therapeutic potential in preventing tumor growth.
Moreover, combining BAPTA_AM treatment with EGFR inhibitors could help, on one hand to reduce drug doses and thus elevated toxicity, and on the other hand, to overcome EGFR acquired resistance to EGFR-TKIs.
This kind of treatment fits into the frame of more accurate therapy, along the lines of constantly developing personalized medicine.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/10/1588/s1, Figure S1: Western blot analysis of EGFR pathway activation in HepG2, HUH-7, HUH-6, and Hep3B cell lines, Figure S2: Western blot analysis of HepG2, HUH-7, HUH-6, and Hep3B starved cell lines treated with GEF IC50 or AZ IC50 (as indicated in Table 1) (DMSO as control) for 3 h before stimulation with 100 ng/mL of EGF for 30 min, Figure S3: A and B: Western blot panels of HepG2, HUH-7, HUH-6, and Hep3B starved cell lines stimulated with 100 ng/mL of EGF for 30 min before and during treatment with GEF or AZ IC50 (as indicated in Table 1) (DMSO as control). Treatments were performed for 30 min, 3 h and 6 h, Figure S4: Starved HUH-7 cells (T0) were left untreated (/) (0% FBS as CTR) or treated with 100 ng/mL EGF, 2 mM EDTA, 0.5% DMSO, or combined compounds (as indicated in the figures). The cell signaling cascade was analyzed by western blot after 6 h and 24 h, Figure S5: HUH-7 cells treated with EDTA or EGTA for 24 h with or without EGF were analyzed by western blot, Figure S6: A and B: Starved HUH-7 and HUH-6 cells (T0) were left untreated (as CTR) or treated with 2 mM EDTA or 10 µM BAPTA_AM with or without 100 ng/mL EGF. The cell signaling cascade was analyzed by western blot after 6 h and 24 h, Figure S7: A and B: Starved HUH-7, HUH-6, HepG2, and Hep3B cells (T0) were left untreated (as CTR) or treated with 10 µM BAPTA_AM. After 30 min, 40 µM MG132 were added for a further 30 min. 100 ng/mL EGF were added for a total time of 6 h before cells harvesting.