Hochuekkito (TJ-41), a Kampo Formula, Ameliorates Cachexia Induced by Colon 26 Adenocarcinoma in Mice

Cachexia, a major cause of cancer-related death, is characterized by depletion of muscle and fat tissues, anorexia, asthenia, and hypoglycemia. Recent studies indicate that secretions of proinflammatory cytokines such as interleukin-6 (IL-6) play a crucial role in cachexia development, and that these cytokines are secreted from not only cancer cells but also host cells such as macrophages. In this study, we investigated the therapeutic effects of hochuekkito, a Kampo formula, on cachexia induced by colon 26 adenocarcinoma in mice. Hochuekkito treatment did not inhibit tumor growth, but significantly attenuated the reduction in carcass weight, food and water intake, weight of the gastrocnemius muscle and fat tissue around the testes, and decrease of serum triglyceride level compared with controls. Furthermore, hochuekkito treatment significantly reduced serum IL-6 level and IL-6 expression level in macrophages in tissues surrounding the tumor. In vitro studies showed that hochuekkito suppressed the production of IL-6 by THP-1 or RAW264.7 macrophage cells, although it did not affect IL-6 production by colon 26 carcinoma cells. These results suggest that hochuekkito inhibits the production of proinflammatory cytokines, particularly IL-6, by host cells such as macrophages. Therefore, hochuekkito may be a promising anticachectic agent for the treatment of patients with cancer

Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor.

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as "gefitinib-resistant persisters" (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1-all genes involved in stemness-were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia

GRPs were highly enriched for gene expression of stemness, IGF1, and IGF1R.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1, which are stemness genes in PC9 or HCC827 parental cells and GRPs. <b>B.</b> Quantitative RT-PCR was performed with primers specific for IGF1 and IGF1R in PC9 or HCC827 parental cells and GRPs. Data were normalized to actin expression. *p<0.01, **p<0.001, ***p<0.0001.</p

Tumor incidence of parental PC9 cells and normoxic and hypoxic GRPs transplanted into NOG mice.

<p>GRPs; Gefitinib resistant persisters.</p><p>NOG mice; NOD/Shi-scid/IL-2Rcnull (NOG) mice.</p>#<p>Tumor incidence was increased significantly in hypoxic GRPs group as compared with parental cell group with 1×10¹ cells/injection, p<0.05.</p><p>To evaluate the <i>in vivo</i> tumorigenic potential, 1×10 cells or 1×10² cells of parental PC9 cells or normoxic and hypoxic PC9 GRPs were mixed with Matrigel and injected into both flanks of NOG mice. Tumor formation was evaluated 33 days after injection.</p

Hypoxia regulates IGF1 expression through HIF1α, and the inhibition of HIF1α or IGF1R decreased CD133- and Oct4-positive GRPs under hypoxia.

<p><b>A.</b> HIF1α expression was suppressed in PC9 or HCC827 hypoxic GRPs by treatment with 50 µM, 100 µM, and 200 µM YC-1 in Lab-Tek chamber slides. Immunofluorescence staining for IGF1, phosphorylated IGF1R (pIGF1R), CD133, and Oct4 was then performed. The numbers of IGF1-, pIGF1R-, CD133-, and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields from each experiment. **p<0.001. <b>B.</b> PC9 or HCC827 cells were incubated with 1 or 2 µM gefitinib in the presence or absence of 0.01 µM, 0.1 µM, or 1 µM of the IGF1R inhibitor AEW541 under hypoxic conditions for 72 h in Lab-Tek chamber slides. Immunofluorescence staining for CD133 and Oct4 was then performed. The numbers of CD133- and Oct4-positive cells were counted, and the ratio was calculated in five fields for each experiment. **p<0.001. <b>C.</b> PC9 or HCC827 cells were plated in 10-cm plates and allowed to adhere for 24 h. Cells were then incubated with 1 or 2 µM gefitinib in the presence or absence of 0.01 µM, 0.1 µM, or 1 µM of AEW541 under hypoxic conditions for 18 or 11 days. The numbers of colonies were then counted.</p

IGF1R was phosphorylated on hypoxic GRPs, and knockdown of IGF1 decreased the population of CD133- and Oct4-positive hypoxic GRPs.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for IGF1 in PC9 or HCC827 parental cells, GRPs, and hypoxic GRPs. Data were normalized to actin expression. **p<0.001. <b>B.</b> PC9 cells, grown on Lab-Tek chamber slides with or without 1 µM gefitinib and under normoxic or hypoxic conditions for 72 h, fixed, and incubated with the primary antibodies against phosphorylated IGF1R (pIGF1R) and then with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained using an Axioplan 2 system imaging with AxioVision software. Images used to compare PC9 parental cells, GRPs, and hypoxic GRPs were acquired with the same instrument settings and exposure times, and were processed similarly. The number of pIGF1R-positive cells was counted, and the ratio of these cells was calculated in five fields in each experiment. **p<0.001. <b>C.</b> IGF1 expression was knocked down in PC9 or HCC827 hypoxic GRPs by using small interfering RNA (siRNA) in Lab-Tek chamber slides. Immunofluorescence staining for pIGF1R, CD133, or Oct4 was then performed. Two specific siRNAs and one non-specific control were used. The numbers of pIGF1R-, CD133- and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields for each experiment. **p<0.001, *p<0.01.</p

Sphere-forming ability of GRPs was upregulated and hypoxia increased this capacity.

<p>PC9 parental cells, GRPs, and hypoxic GRPs were prepared at densities of 2.5×10³ cells per 2 mL per well in serum-free media supplemented with growth factors and seeded into 6-well ultra-low attachment plates. PC9 parental cells and GRPs were incubated under normoxic conditions, while PC9 hypoxic GRPs were grown under hypoxic conditions. Culture medium was fed every 3 days. The number and size of spheres were recorded and immunofluorescence was performed 7 days after the start of the culture period. Spheres were fixed and incubated with primary antibodies against CD133, Oct4, or phosphorylated IGF1R (pIGF1R), and then with secondary antibody labeled with Alexa Fluor 594 goat anti-mouse IgG (red) or Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained on an Axioplan 2 imaging system with AxioVision software. <b>A.</b> The number of spheres was significantly increased in PC9 GRPs compared to in parental cells, and was further increased in PC9 hypoxic GRPs. *p<0.01, # p<0.05. <b>B.</b> Sphere size of PC9 hypoxic GRPs was significantly greater than that of parental cells. # p<0.05. <b>C.</b> Immunofluorescent images of control cells of PC9 (left) and spheres of GRPs (right) for CD133, Oct4, and pIGF1R.</p