Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 18;11(10):1588.
doi: 10.3390/cancers11101588.

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

Teresa Maria Elisa Modica  1   2 , Francesco Dituri  3 , Serena Mancarella  4 , Claudio Pisano  5 , Isabel Fabregat  6   7   8 , Gianluigi Giannelli  9
Affiliations

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

Teresa Maria Elisa Modica et al. Cancers (Basel). .

Abstract

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.

Keywords: AZD9291; BAPTA_AM; EGFR degradation; HCC; calcium ions.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(A) Western blot analysis of EGFR pathway activation in HepG2, HUH-7, HUH-6, and Hep3B cell lines. (B) Densitometric analysis calculated by image lab software of the western blot shown in 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 (***); p value < 0.0001 (****).
Figure 2
Figure 2
(A) 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. Panel (B) shows the densitometric analysis calculated by image lab software of the western blot shown in 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 (***).
Figure 3
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.
Figure 4
Figure 4
Five days proliferation assay of (A) HepG2, (B) HUH-7, (C) HUH-6, and (D) Hep3B cell lines. 10% FBS culture medium was added with GEF IC50 or AZ IC50 (as indicated in Table 1), DMSO as control. Data are plotted in the graph as normalized by DMSO.
Figure 5
Figure 5
Starved HUH-7 (A) and HUH-6 (B) cell lines (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 graphs) for 24 h. After 24 h of treatment, medium was replaced and cells were maintained in culture for a further 48 h in serum-free medium (0% FBS) with or without EGF, on the basis of the previous treatment. The proliferative capability of the cells was evaluated after a total time of 72 h. p value < 0.05 (*).
Figure 6
Figure 6
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 (A,B) and 24 h (B).
Figure 7
Figure 7
HUH-7 not starved (10% FBS (A)) or starved (SF medium (B)) cells were left untreated (medium as CTR) or treated with 2 mM EGTA for 48 h in the presence or absence of CaCl2. The cell signaling cascade was analyzed by western blot after 24 h (C). p value < 0.05 (*); p value < 0.01 (**).
Figure 8
Figure 8
HUH-7 not starved (10% FBS (A)) or starved (SF medium (B)) cells were left untreated (medium as CTR) or treated with 2 mM EGTA for 48 h in the presence or absence of GEF or AZ.
Figure 9
Figure 9
Starved HUH-7 (A,B) and HUH-6 cells (C) (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 10
Figure 10
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.
Figure 11
Figure 11
48 h proliferation assay of (A) HUH-7, (B) HUH-6, (C) Hep3B and (D) HepG2 cell lines. After 2 h starvation (in 0% FBS medium) cells were treated or not in 10% FBS medium with BAPTA_AM for 1 h. After 1 h, 0.5 × GEF, AZ or DMSO (as CTR) was added to the medium for 48 h. p value < 0.05 (*); p value < 0.01 (**).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Llovet J.M., Beaugrand M. Hepatocellular carcinoma: Present status and future prospects. J. Hepatol. 2003;38:136–149. doi: 10.1016/S0168-8278(02)00432-4. - DOI - PubMed
    1. Parkin D.M., Bray F., Ferlay J., Pisani P. Global cancer statistics. CA Cancer J. Clin. 2005;55:74–108. doi: 10.3322/canjclin.55.2.74. - DOI - PubMed
    1. Larsson S.C., Wolk A. Overweight, obesity and risk of liver cancer: A meta-analysis of cohort studies. Br. J. Cancer. 2007;97:1005–1008. doi: 10.1038/sj.bjc.6603932. - DOI - PMC - PubMed
    1. Villanueva A., Llovet J.M. Targeted therapies for hepatocellular carcinoma. Gastroenterology. 2011;140:1410–1426. doi: 10.1053/j.gastro.2011.03.006. - DOI - PMC - PubMed
    1. Villanueva A., Newell P., Chiang D., Friedman S., Llovet J.M. Genomics and Signaling Pathways in Hepatocellular Carcinoma. Semin. Liver Dis. 2007;27:55–76. doi: 10.1055/s-2006-960171. - DOI - PubMed