Mitochondrial complex I inhibitors suppress tumor growth through concomitant acidification of the intra- and extracellular environment

Abstract

The disruption of the tumor microenvironment (TME) is a promising anti-cancer strategy, but its effective targeting for solid tumors remains unknown. Here, we investigated the anti-cancer activity of the mitochondrial complex I inhibitor intervenolin (ITV), which modulates the TME independent of energy depletion. By modulating lactate metabolism, ITV induced the concomitant acidification of the intra- and extracellular environment, which synergistically suppressed S6K1 activity in cancer cells through protein phosphatase-2A-mediated dephosphorylation via G-protein-coupled receptor(s). Other complex I inhibitors including metformin and rotenone were also found to exert the same effect through an energy depletion-independent manner as ITV. In mouse and patient-derived xenograft models, ITV was found to suppress tumor growth and its mode of action was further confirmed. The TME is usually acidic owing to glycolytic cancer cell metabolism, and this condition is more susceptible to complex I inhibitors. Thus, we have demonstrated a potential treatment strategy for solid tumors.

Keywords: Cancer; Cell biology; Microenvironment.

Graphical abstract

Figure 1

Intervenolin (ITV) exhibits anti-cancer activity that is potentiated in co-culture conditions (A) Structure of ITV. (B) Growth of MKN-74 cells cultured alone (Mono) or with Hs738 (Co) for 3 days at the indicated ITV concentrations were measured using GFP fluorescence intensity and expressed as percentage relative to that of cells without ITV treatment (n = 3; ∗p < 0.005, ∗∗p < 0.001 versus mono). (C) Growth of MKN-74 cells upon treatment with ITV CM, Ctrl CM, and their respective controls (DMEM + ITV and Ctrl CM + ITV) for 3 days (∗p < 0.001 versus Ctrl CM+ ITV). For each condition, 1 μg/mL ITV was used. (D) Growth of cancer cell lines MKN-1, -B, and −74 (n = 3). (E) Formalin-fixed, paraffin-embedded tumor sections from mice inoculated with MKN-74 and Hs738 cells treated with the vehicle or 12.5 mg/kg ITV were stained with Masson's trichrome stain and analyzed using immunohistochemistry with antibodies against the indicated proteins. Scale bar, 100 μm (n = 5). (F) Percentages of Ki-67-positive cells in the high-power field of tumor sections (n = 5; ∗p < 0.01). (G) MKN-74 cells were cultured alone (Mono) or with fibroblast cells isolated from mice tumor tissues (+nude FB74-1,2) (Co) for 3 days in the presence of ITV at the indicated concentrations (n = 3; ∗p < 0.0005 versus mono). Data are presented as the mean ± SD and were analyzed using two-sided Student's t test.

Figure 2

Intervenolin (ITV) inhibits mitochondrial complex I activity and induces metabolic shift in cancer cells (A) Results of seahorse flux analyzer assays showing the OCR of MKN-74 cells. Assay buffer (Ctrl) or the indicated concentrations of ITV were introduced at 24 min; FCCP, 64 min; and rotenone (ROT) and antimycin A (AA), 88 min. (B) Activity of the mitochondrial complex I as measured using an NADH assay. Reduction rate of NADH that normalized by non-treated value was shown as complex I activity. The activity of mitochondria without compounds was defined as 100%. (C) Seahorse flux analyzer assay results showing the extracellular acidification rate (ECAR) in MKN-74 cells as in (A). (D) Energy map of MKN-74 cells based on OCR and ECAR values. The status of the metabolic pathways is classified as aerobic, glycolytic, energetic, or quiescent. (E–G). Intracellular metabolite concentrations were determined using mass spectrometric analysis (E and F). Relative amounts of the indicated metabolites in MKN-74 cells treated by 1 μg/mL ITV, 1 μg/mL AS-1936 (AS), or 0.01 μg/mL rotenone (ROT) for 3 h are shown. B.D., below detection limit (n = 3; ∗p < 0.05 versus Ctrl, ∗∗p < 0.005 versus Ctrl). (G) Relative amounts of the indicated metabolites in MKN-74 cells treated by ITV for 3 h are shown (n = 3; ∗p < 0.05 versus non-treated cells). Data are presented as the mean ± SD (n = 3) and normalized according to the baseline and analyzed using two-sided Student's t -test.

Figure 3

Complex I inhibition and low extracellular pH synergistically suppress S6K1 (A) MTT assay results showing the growth of MKN-B and MKN-74 cells cultured with intervenolin (ITV; 1 μg/mL), low pH medium (pH), low pH medium with ITV (1 μg/mL) (pH/ITV), or ITV CM. Cell viability grown at DMEM without NaOH was defined as 100% (n = 3; ∗p < 0.05). (B–D) Western blot analysis showing the phosphorylation levels of S6K1 (T389) in cancer cells with the following culture conditions: (B and D) MKN-B and MKN-74 cells treated with ITV (1 μg/mL), metformin (MET, 1 mg/mL), rotenone (ROT, 0.01 μg/mL), or AS-1936 (1 μg/mL) in DMEM supplemented 1% D-FBS (−), 10 mM sodium lactate (Lac Na), 10 mM lactic acid (Lac acid), or low pH medium (pH) for 3 h. Relative intensities of phospho-S6K1/total S6K1 ratio (versus ctrl) were analyzed by ImageJ and shown. (C) MKN-B and MKN-74 cells cultured in Ctrl CM or ITV CM for 3 h. Data were analyzed using two-sided Student's t test.

Figure 4

Suppression of S6K1 by intervenolin (ITV) under acidic conditions is dependent on PP2A (A–D) Western blot analysis showing the phosphorylation levels of S6K1 (T389) in cancer cells with the following culture conditions: (A) MKN-B and MKN-74 cells cultured in DMEM supplemented with 1% D-FBS (DMEM), ITV CM, or low-pH medium containing ITV (1 μg/mL) (pH/ITV) for 3 h and added with NaOH (Na, 0.1 mM) or calyculin A (Cal, 2 nM) for further 30 min incubation; (B and C) MKN-B and MKN-74 cells cultured in DMEM supplemented 1% D-FBS (DMEM), Ctrl CM, ITV CM, or temsirolimus (25 μM) and added with calyculin A (2 nM) for the indicated time; (D) MKN-B and MKN-74 cells treated with non-target siRNA (siCtrl) or siRNA for PPP2CA and PPP2CB (PPP2CA/B) and cultured in DMEM supplemented 1% D-FBS (Ctrl) or the medium containing 10 mM lactate and ITV (1 μg/mL; ITV/Lac) for 3 h.

Figure 5

GPR132 is important for intervenolin (ITV) activity, and complex I inhibition leads to intracellular acidification (A) Relative mRNA expression levels of GPR68 and GPR132 in MKN-B and MKN-74 (versus MKN-74) as measured using qPCR (n = 3). (B) Relative mRNA expression levels of GPR132 in MKN-74 parent cells (parent) and GPR132-knockout cells (GPR132-KO). (C) Parent and GPR132-KO cells were cultured alone (Mono) or with Hs738 (Co) for 3 days with the indicated concentrations of ITV. Cell growth was determined by measuring GFP fluorescence intensity (n = 3; ∗p < 0.05, ∗∗p < 0.005 versus mono). (D) Parent and GPR132-KO cells were cultured in the medium containing 8 mM lactate with the indicated concentrations of ITV for 1 h. Western blot analysis shows the phosphorylation levels of S6K1 (T389) in cancer cells. (E and F) Intracellular pH levels of cancer cells measured using pHrodo Red fluorescence intensity normalized to that of GFP. MKN-B and MKN-74 cells were treated with 1 μg/mL ITV or 0.1 μg/mL nigericin (NGC) for 3 h. Cells not treated with pHrodo Red served as the negative control group (Nega). Scale bar, 100 μm (n = 3; ∗p < 0.005 versus ctrl). (G) MKN-B and MKN-74 cells cultured in the medium containing 10 mM lactate with the indicated concentrations of nigericin for 3 h. Western blot analysis shows phosphorylation levels of S6K1 (T389) in cancer cells. Data are presented as the mean ± SD and were analyzed using two-sided Student's t test.

Figure 6

Intervenolin (ITV) suppresses intratumor S6K1 phosphorylation and elevates intratumor lactate levels in MKN-74-inoculated mouse xenograft models (A–C) Western blot analysis showing the phosphorylation levels of S6K1 (T389), RPS6 (S235/236), and AMPK (T172) in tumor tissues from mice treated with ITV, rotenone (ROT), or metformin (MET) at the indicated doses. Signal intensities of the bands are shown in the bar graph (n = 3; ∗p < 0.005 versus vehicle). (D) Formalin-fixed, paraffin-embedded tumor sections from mice treated with the vehicle, ITV, ROT, or MET were analyzed by immunohistochemistry using antibodies against phospho-S6K1 (T389). Scale bar, 100 μm (n = 3). (E) Lactate levels in tumor tissues from mice treated with the vehicle, ITV, ROT, or MET (n = 3; ∗p < 0.005 versus vehicle). (F) S6K1 phosphorylation and lactate levels in tumor tissues from mice treated with the vehicle, ITV, ROT, or MET are significantly inversely correlated. r represents Pearson's correlation coefficient. Data are presented as the mean ± SD and were analyzed using two-sided Student's t test.

Figure 7

Intervenolin (ITV) suppresses tumor growth in mouse PDX models (A) Mice treated with ITV (12.5 mg/kg; n = 6) did not show any weight loss compared with those with the vehicle (n = 7). (B) Relative tumor volumes (∗p < 0.005, ∗∗p < 0.01 versus vehicle) at 17 days from ITV injection. ITV was injected on the days indicated by black triangles. (C) Immunohistochemistry analysis of tumor sections from mice treated with the vehicle or ITV. Scale bar, 100 μm. (D–F) Percentages of BrdU-, S6K1-, and p-S6-positive cells in the tumor sections (∗p < 0.05). (G) Proposed mechanism of ITV anti-cancer activity. Data are presented as the mean ± SD and were analyzed using two-sided Student's t test.