Boosting nutrient starvation-dominated cancer therapy through curcumin-augmented mitochondrial Ca2+ overload and obatoclax-mediated autophagy inhibition as supported by a novel nano-modulator GO-Alg@CaP/CO

Abstract

Background: By hindering energy supply pathway for cancer cells, an alternative therapeutic strategy modality is put forward: tumor starvation therapy. And yet only in this blockade of glucose supply which is far from enough to result in sheer apoptosis of cancer cells.

Results: In an effort to boost nutrient starvation-dominated cancer therapy, here a novel mitochondrial Ca²⁺ modulator Alg@CaP were tailor-made for the immobilization of Glucose oxidase for depriving the intra-tumoral glucose, followed by the loading of Curcumin to augment mitochondrial Ca²⁺ overload to maximize the therapeutic efficiency of cancer starvation therapy via mitochondrial dysfunctions. Also, autophagy inhibitors Obatoclax were synchronously incorporated in this nano-modulator to highlight autophagy inhibition.

Conclusion: Here, a promising complementary modality for the trebling additive efficacy of starvation therapy was described for cutting off the existing energy sources in starvation therapy through Curcumin-augmented mitochondrial Ca²⁺ overload and Obatoclax-mediated autophagy inhibition.

Keywords: Autophagy; Cancer metabolism; Complementary modality; Curcumin; Mitochondrial Ca2+ overload; Starvation therapy.

Scheme 1

A Schematic diagram of the novel nano-modulator GO-Alg@CaP/CO. B Schematic illustration of the promising complementary modality for the trebling additive efficacy of starvation therapy which was described for cutting off the existing energy sources in starvation therapy through curcumin-augmented mitochondrial Ca²⁺ overload and obatoclax-mediated autophagy inhibition

Fig. 1

Particle sizes of A CaP, B Alg@CaP and C GO-Alg@CaP/CO. These insets showed the images of the corresponding aqueous solution. Transmission electron microscopy (TEM) of D CaP, E Alg@CaP and F GO-Alg@CaP/CO (scale bars were 200 nm). G The standard content curves of GO based on BCA assays. H Variation of glucose concentration and I pH value in glucose solution (1 mg/mL) after the addition of GO, GO-Alg and GO-Alg@CaP/CO with the identical GO concentration (1 mg/mL)

Fig. 2

Disruption of intramitochondrial Ca²⁺ homeostasis A intracellular Ca²⁺ production and mitochondrial Ca²⁺ concentrations of 4T1 cells treated with different preparations for 6 h. Scale bars were 50 μm. B–E Flow cytometry results of 4T1 cells incubated with GO-Alg@CaP and GO-Alg@CaP/C. Data are mean ± SD, n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 3

Biodistribution of Ca²⁺ in mitochondria in 4T1 cells after incubation with different preparations for 6 h. The nucleus and mitochondria were stained with Hoechst 33342 and Mito-Green, respectively. The scale bar was 10 μm

Fig. 4

A P62, LC3-I, and LC3-II expression in 4T1 cells with a western-blotting analysis under different groups. Matching gray-scale analysis of B LC3-II/LC3-I and C P62. D TEM images of autophagosome. Red arrows and circles implied the autophagosome. Data are mean ± SD, n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001 vs control. The scale bar was 5 μm

Fig. 5

A Relative viability of 4T1 cancer cells cultured with blank carriers (Alg@CaP) at different concentrations. Relative viability of 4T1 cancer cells cultured with different treatments. B GO and GO-Alg@CaP, C Cur and Alg@CaP/C, and D obatoclax and Alg@CaP/O. E Relative viability of 4T1 cancer cells cultured with different preparations at desired concentrations for 24 h. F Intracellular ATP content analysis of 4T1 cancer cells cultured with different preparations at desired concentrations for 48 h. G Apoptosis assays of 4T1 cells in different groups with a flow cytometric analysis. Data are mean ± SD, n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control

Fig. 6

A In vivo fluorescence images of 4T1 tumor-bearing mice that were intravenously injected with Alg@CaP/I at different time points and B ex vivo fluorescence images of major organs and tumors at 24 h post injection. C Histogram analysis of relative average radiant efficiency of liver, lungs, and tumor. D TUNEL-stained tumor slices collected from 4T1 tumor-bearing mice after treatments for 15 days. The scale bar was 50 μm. E Blood analysis of mice at 15 days after various treatments. F Relative volume changes and G mice body weight changes in different groups. Data are mean ± SD, n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control

Fig. 7

A Representative flow cytometric analysis (left) and quantification (right) of CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells in the tumor. B Representative flow cytometric analysis gating on CD3⁺ cells (left) and relative quantification (right) of CD4⁺CD8⁺ T cells and CD3⁺cells. Data are mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001