Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells

It has long been known that the gap junction is down-regulated in many tumours. One of the downregulation mechanisms is the translocation of connexin, a gap junction protein, from cell membrane into cytoplasm, nucleus, or Golgi apparatus. Interestingly, as tumours progress and reinforce their malignant phenotype, the amount of aberrantly-localised connexin increases in different malignant tumours including oesophageal squamous cell carcinoma, thus suggesting that such an aberrantly-localised connexin should be oncogenic, although gap junctional connexins are often tumour-suppressive. To define the dual roles of connexin in head and neck squamous cell carcinoma (HNSCC), we introduced the wild-type connexin26 (wtCx26) or the mutant Cx26 (icCx26) gene, the product of which carries the amino acid sequence AKKFF, an endoplasmic reticulum-Golgi retention signal, at the C-terminus and is not sorted to cell membrane, into the human FaDu hypopharyngeal cancer cell line that had severely impaired the expression of connexin during carcinogenesis. wtCx26 protein was trafficked to the cell membrane and formed gap junction, which successfully exerted cell-cell communication. On the other hand, the icCx26 protein was co-localised with a Golgi marker, as revealed by immunofluorescence, and thus was retained on the way to the cell membrane. While the forced expression of wtCx26 suppressed both cell proliferation in vitro and tumorigenicity in mice in vivo, icCx26 significantly enhanced both cell proliferation and tumorigenicity compared with the mock control clones, indicating that an excessive accumulation of connexin protein in intracellular domains should be involved in cancer progression and that restoration of proper subcellular sorting of connexin might be a therapeutic strategy to control HNSCC

Intrinsic Oncogenic Function of Intracellular Connexin26 Protein in Head and Neck Squamous Cell Carcinoma Cells

It has long been known that the gap junction is down-regulated in many tumours. One of the downregulation mechanisms is the translocation of connexin, a gap junction protein, from cell membrane into cytoplasm, nucleus, or Golgi apparatus. Interestingly, as tumours progress and reinforce their malignant phenotype, the amount of aberrantly-localised connexin increases in different malignant tumours including oesophageal squamous cell carcinoma, thus suggesting that such an aberrantly-localised connexin should be oncogenic, although gap junctional connexins are often tumour-suppressive. To define the dual roles of connexin in head and neck squamous cell carcinoma (HNSCC), we introduced the wild-type connexin26 (wtCx26) or the mutant Cx26 (icCx26) gene, the product of which carries the amino acid sequence AKKFF, an endoplasmic reticulum-Golgi retention signal, at the C-terminus and is not sorted to cell membrane, into the human FaDu hypopharyngeal cancer cell line that had severely impaired the expression of connexin during carcinogenesis. wtCx26 protein was trafficked to the cell membrane and formed gap junction, which successfully exerted cell-cell communication. On the other hand, the icCx26 protein was co-localised with a Golgi marker, as revealed by immunofluorescence, and thus was retained on the way to the cell membrane. While the forced expression of wtCx26 suppressed both cell proliferation in vitro and tumorigenicity in mice in vivo, icCx26 significantly enhanced both cell proliferation and tumorigenicity compared with the mock control clones, indicating that an excessive accumulation of connexin protein in intracellular domains should be involved in cancer progression and that restoration of proper subcellular sorting of connexin might be a therapeutic strategy to control HNSCC

The Apoptotic Effect of HIF-1α Inhibition Combined with Glucose plus Insulin Treatment on Gastric Cancer under Hypoxic Conditions

<div><p>Gastric cancer grows under a hypoxic environment. HIF-1α is known to play an important role in controlling the production of reactive oxygen species (ROS) in the mitochondria under hypoxic conditions. We previously established HIF-1α knockdown (KD) cells and control (SC) cells in the 58As9 gastric cancer cell line. In this study, we revealed that KD cells, but not SC cells, induced apoptosis under conditions of hypoxia (1% O₂) due to excessive production of ROS. A quantitative RT-PCR analysis demonstrated that the expressions of ten genes, which are involved in the control mechanisms of ROS (including the Warburg effect, mitophagy, electron transport chain [ETC] modification and ROS scavenging), were regulated by HIF-1α. Moreover, the promotion of glucose uptake by glucose plus insulin (GI) treatment enhanced the apoptotic effect, which was accompanied by further ROS production in hypoxic KD cells. A Western blot analysis showed that the membranous expression of GLUT1 in KD cells was elevated by glucose and/or insulin treatments, indicating that the GI-induced glucose uptake is mediated by the increased translocation of GLUT1 on the cell membrane. Finally, the anti-tumor effect of HIF-1α knockdown (KD) plus GI was evaluated using a tumor xenograft model, where a hypoxic environment naturally exists. As a result, the GI treatment strongly inhibited the growth of the KD tumors whereby cell apoptosis was highly induced in comparison to the control treatment. In contrast, the growth of the SC tumors expressing HIF-1α was not affected by the GI treatment. Taken together, the results suggest that HIF-1α inhibition plus GI may be an ideal therapy, because the apoptosis due to the destruction of ROS homeostasis is specifically induced in gastric cancer that grows under a hypoxic environment, but not in the normal tissue under the aerobic conditions.</p></div

The <i>in vivo</i> effect of HIF-1αknockdown plus GI treatment on tumor xenografts.

<p>(A) The experimental schedule of glucose (G) or glucose plus insulin (GI) treatment on the tumor xenografts. The initial intraperitoneal (i.p) injection with control PBS or Glucose, GI is indicated by the empty triangle. The i.p injections are indicated by arrows. The tumors harvested on day 12 are indicated by the reticulated triangle. (B) The Western blot analysis of HIF-1α in the xenografts of the SC cells (SC tumor) and KD cells (KD tumor). (C) Representative images of the SC or KD tumors treated with PBS, Glucose or GI. The treated tumors were designated as SC-PBS, SC-Glucose, SC-GI, KD-PBS, KD-Glucose and KD-GI. (D) The size of the 6 tumors on day 1 to day 12 was plotted on the graph as indicated. (E) The immunohistochemical analysis of cleaved caspase 3 was performed using six tumors of SC-PBS, SC-Glucose, SC-GI, KD-PBS, KD-Glucose and KD-GI. (F) The mean positive rate of cleaved caspase 3 in 6 tumors was estimated and presented in the graph. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001.</p

Primer sequences for ten genes involved in regulating ROS production.

<p>Primer sequences for ten genes involved in regulating ROS production.</p

The anti-tumor effect of GI treatment on HIF-1α knockdown KD cells under hypoxic conditions.

<p>(A) HIF-1α plays a central role in controlling the ROS level in the SC cells under hypoxia. HIF-1α induces the gene expression of ten specific genes under hypoxia (indicated by red letters). (B) The apoptotic effect induced by HIF-1α knockdown plus GI treatment in the KD cells under hypoxia. The hypoxia-induced expression of the 10 genes is reduced by HIF-1α knockdown (indicated by gray letters). NC: Nucleus, MC: Mitochondria, GL: Glycolysis, WE: Warburg effect, MP: Mitophagy, IR: Insulin receptor.</p

The examination of glucose uptake in SC and KD cells with or without insulin treatment.

<p>(A), (B) The 2DG uptake level in SC cells or KD cells with or without insulin treatment was evaluated under normoxia (A) and hypoxia (B). (C) The Western blot analysis of membranous GLUT1 (54 kDa) expression in the KD cells with control, high glucose and/or insulin treatments under both normoxia and hypoxia as indicated. ***: p<0.001.</p

The effect of GI treatment on cell death and ROS production.

<p>The FI of the cell death rate was determined as the death ratio of hypoxia/normoxia. The cell death rate in PBS-treated (control), high glucose and/or insulin-treated SC cells (A) and KD cells (B) is plotted. The FI value is presented on the bottom. The cell death rate under hypoxic conditions in the control treatment was compared with that in high glucose, insulin and GI treatment. (C) The intracellular ROS level in the control-treated, high glucose and/or insulin-treated KD cells are plotted on the graph. The ROS levels in the KD cells treated with the control treatment were compared between normoxia (black bars) and hypoxia (white bars). The ROS level in the hypoxic KD cells with control treatment was further compared with that with high glucose, insulin and GI treatment. ns: not significant, *: p<0.05, **: p<0.01, ***: p<0.001.</p