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Back to Journal »International Journal of Nanomedicine» Volume 16

PI3K/mTOR dual inhibitor BEZ235 and nanoemulsion paclitaxel can synergistically inhibit the growth of drug-resistant colon cancer by reducing multidrug resistance and promoting apoptosis

Authors: Hu Y, Zhang Kun, Zhu X, Zheng X, Wang C, Niu X, Jiang Tao, Ji X, Zhao Wen, Pang Li, Qi Y, Li Fei, Li Li, Xu Zhen, Gu Wei, Zou Hong

Published on March 15, 2021, the 2021 volume: 16 pages 2173-2186

DOI https://doi.org/10.2147/IJN.S290731

Single anonymous peer review

Editor who approved for publication: Dr. Farooq A. Shiekh

Hu Yali, 1, 2, * Zhang Kunpeng, 1, * Zhu ​​Xingyao, 1, * Zheng Xiuyan, 1 Super King, 1 Mavericks, 1 Teng Jiang, 1 Xinhua Ji, 1 Zhao Weilin, 1 Pang Lijuan, 1 Yan Qi, 1 Feng Li, 1,3 Li Li,4 Xu Zhiping,4 Gu Wenyi,4 Zou Hong1 1Department of Pathology, The First Affiliated Hospital of Shihezi University School of Medicine, Xinjiang Key Laboratory of Endemic Diseases, Ministry of Education, Xinjiang, 832002; 2 Yongcheng People's Hospital Department of Oncology, Henan 476600; 3 Department of Pathology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 10000; 4 Australian Institute of Bioengineering and Nanotechnology, University of Queensland, Queensland, 4072, Australia *These authors contributed equally to this work Gu Wenyi Tel +86 13899528366; +61733464168 Email [email protection]; [email protection] Background: Colon cancer is the number one deadly cancer in men and women worldwide, and drug resistance is the main cause of cancer-related deaths. The combination therapy and drug delivery of nanoparticles has been shown to be effective in overcoming the drug resistance of many cancers. We previously reported that nanoemulsion (NE) loaded paclitaxel (PTX) and BEZ235 can synergistically inhibit the growth of colon cancer cells. Objective: To study whether PTX and BEZ235 loaded with NE can overcome drug resistance and inhibit the growth of drug-resistant colon cancer cells in vitro and in vivo. Methods: CCK8 kit was used to detect the effect of in vitro treatment on cell viability, β-tubulin immunofluorescence staining to detect cell morphology changes, Western blotting to analyze drug resistance-related proteins, and tumor growth test in nude mice. Xenotransplantation of two drug-resistant colon cancer cell lines HCT116-LOHP and HT29-DDP. Results: Both cell lines are sensitive to PTX, but relatively insensitive to BEZ235. The combination of PTX and BEZ235 can synergistically inhibit the proliferation of the two cell lines. Nanoemulsion-loaded PTX (NE-PTX) reduces the IC50 of PTX to about 2/5 of that of free PTX, indicating that NE-PTX has a high inhibitory effect. When NE-PTX is combined with low concentration of BEZ235 (50 nM), IC50 is reduced to about 2/3 of free PTX. In addition, compared with single-drug treatment and control group, NE-PTX+BEZ235 treatment increased cell apoptosis, decreased the expression of Pgp and ABCC1, and decreased tumor weight. These results indicate that the nanoemulsion loaded with PTX+BEZ235 can overcome drug resistance and improve the inhibition of cancer cell proliferation and tumor growth. Conclusion: Therefore, our study provides a possible new method for the treatment of patients with drug-resistant colon cancer. Keywords: colon cancer, nanoemulsion, paclitaxel, BEZ235, drug resistance

Colon cancer is one of the most common malignant tumors in men and women. Most cases of colon cancer are already at an advanced stage when they are diagnosed. 1 Chemotherapy is a common treatment method. The first-line drugs include 5-FU, platinum drugs and EGFR receptor blockers. However, the multi-drug resistance caused by 5-FU and platinum-based drugs is a major challenge for the successful treatment of colon cancer. So far, several mechanisms of multidrug resistance in colon cancer have been reported. These include enhanced efflux caused by high expression of ATP-binding transporters (such as P-glycoprotein, Pgp), decreased intracellular drug concentration, decreased sensitivity of tumor cells to chemotherapeutic drugs, and development of drug resistance-related enzyme activity and content Changes 2–7 About 46% of colorectal cancers have K-ras gene mutations, 8,9 which makes targeted therapy with EGFR receptor blockers (such as cetuximab) ineffective. 1 The incidence of multidrug resistance in colorectal cancer patients has a metastatic rate of more than 90%. 7,8 Existing colorectal cancer chemotherapy drugs have varying degrees of side effects. Therefore, it has important clinical significance to overcome chemotherapy resistance and find new, effective, and low-toxic drugs or approaches.

Paclitaxel (PTX) can effectively promote cancer cell apoptosis and is effective for tumor cells with mutations in the K-ras gene. 10-15 The PI3K/Akt/mTOR signaling pathway is believed to be closely related to the multidrug resistance of human colon cancer. It is reported that blocking the PI3K/AKT signaling pathway can enhance the drug sensitivity of HCT-116/L-OHP resistant cells and reverse the Pgp-mediated multidrug resistance in human colon cancer. 16,17 BEZ235, a PI3K/Akt/mTOR dual pathway inhibitor, is reported to enhance the sensitivity of colon cancer cell line HCT-116 to 5-FU and inhibit the growth of colon cancer stem cells. 18-20 Multi-drug combination therapy is a common strategy to overcome drug resistance. Here, we speculate that the combination of BEZ235 and PTX may act synergistically on multiple targets of colon cancer cells to reduce drug resistance and promote cell apoptosis. Previously, we demonstrated that PTX and PI3K/mTOR inhibitor BEZ235 had a good synergistic inhibitory effect on the growth of colon cancer cells; and the nanoemulsion loaded with a combination of PTX and BEZ235 showed a more significant improvement in inhibitory activity. 21 However, PTX is a highly hydrophobic molecule, which hinders the delivery of the maximum effective dose to patients and limits the clinical therapeutic effect. Their application in cancer treatment is limited by their low solubility and bioavailability. We further encapsulated PTX into nanoemulsion and then mixed it with BEZ235, and proved that the PTX nanoemulsion-BEZ235 combination therapy further significantly inhibited HCT-116 and HT-29 colon cancer cells due to the increased solubility and bioavailability of PTX Growth. 21 Nanoemulsion nanoparticles are composed of oil, surfactant/co-surfactant and water, and the size is in the range of 10-200 nm. If these drugs are soluble in the oil phase to increase the solubility of the drugs in water, they can encapsulate various hydrophobic drugs together. What is important is that the hydrophobic drugs wrapped in nanoemulsions can be effectively absorbed by cancer cells and circulate for a long time in the body. Studies have reported that paclitaxel nanoemulsion can not only reduce the toxicity of the drug, but also prolong the action time of the drug in the rat. 22,23

Here, we hypothesize that the combination of PTX and BEZ235 may act synergistically on multiple targets of colon cancer cells to reduce drug resistance and promote cell apoptosis. However, the effect of the combination of nanoemulsion loaded PTX (NE-PTX) and BEZ235 on drug-resistant colon cancer cells has not been reported yet. Therefore, we aimed to investigate whether the combination of NE-PTX and BEZ235 can show a synergistic inhibitory effect on the growth of drug-resistant colon cancer cells (in vitro and in vivo), and reduce drug toxicity and side effects. The morphological changes and their underlying mechanisms are also analyzed. The results confirmed that the combined use of NE-PTX and BEZ235 can significantly enhance the inhibition of the growth of drug-resistant colon cancer cells, providing great potential for the future clinical application of colon cancer treatment.

Oxaliplatin-resistant (HCT116-LOHP) and cisplatin-resistant (HT29-DDP) human colon cancer cell lines were purchased from Shanghai Jiguang Technology Co., Ltd. (Shanghai, China). Cisplatin and oxaliplatin, which are used to maintain the resistance of the two colon cancers, were purchased from Sigma in the United States. Both BEZ235 and PTX were purchased from Selleck Chemicals, USA. Oxaliplatin and cisplatin were dissolved and stored in saline, while PTX and BEZ235 were dissolved and stored in DMSO. The sources of other cell culture-related reagents are as follows: RPMI 1640 medium (Invitrogen, USA), 0.25% EDTA trypsin (Beijing Soleibao, China), Fetal Bovine Serum (Biolnd, Israel), Penicillin Streptomycin Mixture (Beijing Solei Bao Company, China), DMSO (Beijing Soleibao Company, China), PBS (Shanghai Shenggong Company, China); CCK-8 Cell Proliferation Toxicity Test Kit (Japan Institute of Chemistry) is used to detect cell proliferation; Skeleton-related reagents include anti-β-tubulin antibody, FITC-labeled anti-rabbit IgG (Abeam, USA), propidium iodide (Sigma), cell cycle detection kit purchased from China Unitech; Western blot analysis of antibody and its concentration Pgp (1:1000, CST, US), ABCC1 (1:1000, CST, US), β-actin (1:1000, Abcam, US), Bcl-2 (1:1000, CST, US) .

Paclitaxel is dissolved in DMSO. Then it was mixed with Capryol 90, Tween 20 and propylene glycol (the volume ratio of Tween 20 to propylene glycol was 2:1) on a vortex mixer to form an emulsified mixture. Water was added to the mixture and the mixture was stirred at the highest speed for 5 minutes to obtain a nanoemulsion. The mass ratio of Capryol 90, Tween 20 and propylene glycol to water is 20:35:45. Dynamic light scattering (Nano ZS, Zetasizer, Nano series, Malvern Instruments) is used to measure the average particle size and distribution of paclitaxel nanoemulsions. The stability was studied by measuring its size distribution characteristics in serum-containing medium, PBS and pH5.8 buffer for 6 consecutive days. The prepared paclitaxel nanoemulsion (2 mM) was stored at 4°C for further analysis.

Both cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 5% penicillin/streptomycin. Add cisplatin (1.5 μg/mL) to the culture medium for culturing the HT29DDP cell line, and add oxaliplatin (10 μg/mL) for culturing the HCT116-LOHP cell line to maintain the corresponding resistance of the two drug-resistant cells Phenotype. The cells were cultured in a constant temperature humidified incubator at 37°C and 5% CO2. For subculture, cells were digested with 0.25% trypsin. HCT116-LOHP or HT29DDP cells were seeded in 96-well plates with a cell density of 4⨯103 cells/well. After overnight incubation, the cells attach and grow in the wells. Then, the RPMI 1640 medium was removed and replaced with fresh RPMI-1640 medium containing different concentrations of BEZ235, PTX, NE-PTX or NE-PTX+BEZ235 (50 nM). Among them, the details of mixing 50nM BEZ235 with different concentrations of PTX or NE-PTX medium are as follows. When BEZ235 is added to the cell culture medium, we mix 1mg/mL BEZ235 concentrate with the cell culture medium to obtain a cell culture medium containing 50nM BEZ235. Then add PTX or PTX to the cell culture medium containing 50 nMBEZ235. NE-PTX, obtain cell culture medium containing 100nM PTX or NE-PTX and 50nMBEZ235, and then add cell culture medium containing BEZ235 to dilute to prepare corresponding medium containing 50nmBEZ235 and different concentrations of PTX and NE-PTX, and then classify according to In the experimental group, change the medium, and then observe and confirm. After 48 hours of treatment, add 10 μL of CCK-8 reagent to each well in the dark, wrap the 96-well plate with foil, and put it in the incubator. After 3 hours, use the xMark microplate reader (BIO-RAD, USA) to measure the absorbance OD value at A450nm, and calculate the cell survival rate as follows:

Cell survival rate% = (absorbance OD value of the experimental group-absorbance of the blank group) ÷ (absorbance OD value of the control group-absorbance of the blank group) × 100%

The combination index is calculated according to the following formula to determine the synergy of the combination therapy:

CI <1, = 1 and> 1 indicate synergistic, additive and antagonistic effects, respectively.

Colon cancer HT29DDP and HCT116-LOHP cells were cultured in 6-well plates (2⨯105 cells per well). After 24 hours, the cells were treated with drugs directly added to the culture medium. After 2 hours of treatment, the medium was removed. The cells were fixed with 4% paraformaldehyde, 0.5% Triton X-100 was permeated for 20 minutes, and 3% goat serum was blocked for 30 minutes at room temperature. The cells were then incubated with diluted rabbit anti-human β-tubulin antibody (Abcam, USA) at 4°C overnight. Subsequently, the cells were washed 3 times with PBST and incubated with FITC (green fluorescent) labeled anti-rabbit fluorescent antibody (Abcam, USA) at 37°C for 1 hour. Then, the cells were washed 3 times with PBST and incubated with PI (propidium iodide purchased from Sigma) to stain the nucleus (red fluorescence). Excess PI was removed by washing with PBS. Then fix the cells on a glass slide with anti-fluorescence quencher, then observe and take pictures under a fluorescence microscope.

Cell cycle analysis kit (Biotech Corp) was used to analyze cell apoptosis. HT29DDP and HCT116-LOHP cells are cultured in 6-well plates. The next day, the cells were treated with PTX, BEZ235, or a combination of the two drugs. After 24 hours, 2⨯105-1⨯106 cells were collected by centrifugation and washed with PBS. Subsequently, add 1 mL of DNA staining solution and 10 mL of permeabilization solution to the cells and vortex to mix for 5-10 seconds. Then, the cells were incubated at room temperature for 30 minutes and analyzed by flow cytometry. Apoptotic cells are gated as a sub-G1/G0 population from a single cell.

After treating HT29DDP and HCT116-LOHP cells with different drugs for 24 hours, the cells were washed twice with PBS to extract total protein. A Nano Drop spectrophotometer was used to determine the protein concentration. The proteins were separated by gel electrophoresis (BIO-RAD, USA) and transferred to PVDF membrane. The membrane was blocked at room temperature (5% BSA) for 2 hours, and then incubated with rabbit anti-human Pgp and ABCC1 and mouse anti-human Bcl-2 and β-actin antibodies at 4°C overnight. Then, the membrane was washed at room temperature, treated with horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG antibodies for 2 hours, and exposed to ECL for observation and imaging.

Our animal experiment was approved by the Animal Ethics Committee of the Affiliated Hospital of Shihezi University School of Medicine. According to China's "Guidelines for the Ethical Review of Laboratory Animal Welfare" (GB/T 35892-2018) and the "3Rs" animal welfare and ethical principles, that is, to replace and reduce animals in experiments, and to improve procedures to reduce their hazards. Female BALB/C-nu nude mice were equally divided into two halves (one injected with HT29DDP and the other injected with HCT116-LOHP cells; each injection of 2⨯106 cells in 50 uL). When the tumor size reaches 50-100 mm3, each half of the animals are randomly divided into five groups (PBS control group, PTX group -10 mg/kg, BEZ235 group -20 mg/kg, NE-PTX group-6.6 mg/kg, NE -PTX+BEZ235 group -6.6 mg/kg, NE-PTX+BEZ235-20 mg/kg; 8 mice per group). EDTA is used to prepare drug solutions. The medicine is injected every two days. A single injection of 50ul of tumor. After every 2 days, the longest diameter (a) and shortest diameter (b) of the tumor were measured with vernier calipers, and the body weight of nude mice was measured every 3 days. After three weeks, the mice were euthanized and the tumor volume was measured (V (mm3) = ab2/2).

The data collected from the experimental group and the control group are expressed as mean ± SD. One-way analysis of variance and unpaired Student's t test (GraphPad Prism 6 program) were used to analyze the differences between the groups and determine the significant differences between the experimental group and the control group (two-tailed, P <0.05).

Homogenous NE-PTX is prepared by low-energy spontaneous emulsification. When the oil/emulsifier/water ratio is 20:35:45, a clear and uniform suspension is obtained, and the final concentration of NE-PTX is 2.0 mM. After loading the drug, the particle size and polydispersity index of drug-NE remain unchanged. 21 The dynamic light scattering results in Figure 1A show that NE-PTX has a narrow particle size distribution, a polydispersity index of 0.19, and a similar particle size and PDI in the medium, PBS, and pH 5.8 buffer. In the subsequent 6-day contact measurement (Figure 1B), we found that the distribution and particle size of NE-PTX remained in the same area without significant changes, especially in the medium, indicating that NE-PTX has good stability. Figure 1 The particle size of NE-PTX from DLS analysis. (A) NE-PTX has a narrow particle size distribution and a polydispersity index of 0.19. It has similar particle size and PDI in RPMI 1640 medium, PBS, and pH 5.8 buffer. (B) In the subsequent 6-day contact measurement, the distribution and particle size of NE-PTX remained in the same area without significant changes, especially in the medium, indicating that NE-PTX has good stability. Abbreviations: DLS, dynamic light scattering; NE, nanoemulsion; PTX, paclitaxel.

Figure 1 NE-PTX particle size from DLS analysis. (A) NE-PTX has a narrow particle size distribution and a polydispersity index of 0.19. It has similar particle size and PDI in RPMI 1640 medium, PBS, and pH 5.8 buffer. (B) In the subsequent 6-day contact measurement, the distribution and particle size of NE-PTX remained in the same area without significant changes, especially in the medium, indicating that NE-PTX has good stability.

Abbreviations: DLS, dynamic light scattering; NE, nanoemulsion; PTX, paclitaxel.

In order to study whether the combination of PTX and BEZ235 has a synergistic effect on drug-resistant colon cancer cells, we first studied the inhibitory effect of a single drug. The drug-resistant cell lines HT29DDP and HCT116-LOHP are both sensitive to PTX treatment (IC50 of 34.78 nM and 37.56 nM, respectively (Table 1, Figure 2A)). However, they are not very sensitive to BEZ235 (IC50 of 483.8 nM and 651.9 nM, respectively; Figure 2B). Since BEZ235 is not sensitive to colon cancer drug-resistant cells, whether low concentrations of BEZ235 can enhance the sensitivity of colon cancer drug-resistant cells to PTX is worth exploring. Therefore, both cells were treated with a combination of PTX and 50 nM BEZ235 (9-13 times lower). Compared with PTX alone, the combination therapy reduced the IC50 of PTX in the two cell lines to one third (21.48 nM and 26.42 nM in the HT29DDP and HCT116-LOHP cell lines, respectively; Figure 2C), indicating that BEZ235 can increase the Cells resistant to PTX. The combination indexes of 50nM BEZ235+10nM PTX in HT29DDP and HCT116-LOHP cell lines were 0.86 and 0.92, respectively (Table 2). These data indicate that the combined method of PTX and BEZ235 has a synergistic therapeutic effect on drug-resistant colon cancer. Table 1 The IC50s of BEZ235 and PTX free or loaded with NE in the treatment of drug-resistant colon cancer cells. Figure 2 The dose-dependent inhibitory effect of PTX, BEZ235, NE-PTX and their combinations on the growth of HT29DDP and HCT116-LOHP. CCK8 measurement. A) The CCK8 assay resulted in HT29DDP and HCT116-LOHP cells treated with different concentrations of PTX. (B) CCK8 assay results show two cell lines treated with different concentrations of BEZ235. (C) The combination of different concentrations of PTX and 50nM-BEZ355 improved the sensitivity of the two cell lines to treatment. (D) NE-PTX showed improved inhibition in both cell lines. (E) The combination of NE-PTX and BEZ235 (NE-PTX + BEZ235) can synergistically inhibit the growth of the two cell lines. (F) Control of NE; no obvious toxic effects were observed in both cell lines at doses below 150 nM. Table 2 The combined index (CI) analysis of BEZ235 and PTX/NE-PTX treatment uses the average value of cell viability (%)

Table 1 The IC50 of BEZ235 and PTX free or loaded with NE in the treatment of drug-resistant colon cancer cells

Figure 2 The dose-dependent inhibitory effect of PTX, BEZ235, NE-PTX and their combination on the growth of HT29DDP and HCT116-LOHP, measured by CCK8 assay. (A) CCK8 assay results in HT29DDP and HCT116-LOHP cells treated with different concentrations of PTX. (B) CCK8 assay results show two cell lines treated with different concentrations of BEZ235. (C) The combination of different concentrations of PTX and 50nM-BEZ355 improved the sensitivity of the two cell lines to treatment. (D) NE-PTX showed improved inhibition in both cell lines. (E) The combination of NE-PTX and BEZ235 (NE-PTX + BEZ235) can synergistically inhibit the growth of the two cell lines. (F) Control of NE; no obvious toxic effects were observed in both cell lines at doses below 150 nM.

Table 2 The combined index (CI) analysis of BEZ235 and PTX/NE-PTX treatment uses the average value of cell viability (%)

Nanoemulsions can reduce toxicity and side effects by increasing the solubility and efficacy of chemotherapy drugs. 21 Therefore, we prepared nanoemulsions to deliver PTX to further improve its efficiency. Compared with PTX (free drug) alone, NE-PTX has an increased inhibitory effect on HT29DDP and HCT116-LOHP cells (IC50 is 21.57 nM and 22.77 nM, respectively, which is about 2/5 (40%) times lower than that ) PTX alone; Figure 2D). The combination of NE-PTX and 50 nM BEZ235 further reduced the IC50 of PTX in HT29DDP and HCT116-LOHP cells to 11.84 nM and 14.78 nM, respectively (Figure 2E). These IC50s are about 1/3 times lower than that of PTX alone, and 1/2 times lower than that of PTX combined with 50 nM BEZ235, indicating that the combined use of NE-PTX and low-concentration BEZ235 also exhibits a more obvious sensitization effect. The combination index of 50nM BEZ235+10nM NE-PTX in HT29DDP and HCT116-LOHP cell lines were 0.79 and 0.88, respectively (Table 2), indicating that the combination of PTX-loaded nanoemulsion and low concentration of BEZ235 can synergistically inhibit the growth of drug-resistant colon cancer cell. A significant reduction in the dose of chemotherapy drugs will significantly reduce their toxicity and side effects, because NE alone does not exhibit too much cytotoxicity, even at 120 nM (Figure 2F).

Except for the significant cell death after 24 hours of treatment (Figure 3A), cell morphological changes were observed in both HT29DDP and HCT116-LOHP cells. Using β-tubulin staining (green fluorescence), we can see that the round cells in the control become more like fibroblasts in the processed sample. The red fluorescence showed that the nucleus was stained by PI, and the morphological changes of the PTX+BEZ235 group were more obvious (Figure 3B). The morphological change is the result of BEZ235 treatment, which is consistent with the previous report. 21 Figure 3 Cell number and morphological changes after treatment with BEZ235 and PTX. (A) Bright field image of cell death induced by PTX and BEZ235 alone or in combination. Compared with the control group, a significant decrease in the number of cells was observed in the treatment group. The image was taken with an Olympus IX51 fluorescence microscope at 100x magnification. (B) The morphological changes of cells treated with PTX or BEZ235 or both for 2 hours. Using β-tubulin staining (green fluorescence) and PI staining (red fluorescence) cell nuclei, observe the morphological changes from round cells in the control group to fibroblast-shaped cells in the treatment group. These images were taken with an Olympus IX51 fluorescence microscope at 400x magnification.

Figure 3 Cell number and morphological changes after BEZ235 and PTX treatment. (A) Bright field image of cell death induced by PTX and BEZ235 alone or in combination. Compared with the control group, a significant decrease in the number of cells was observed in the treatment group. The image was taken with an Olympus IX51 fluorescence microscope at 100x magnification. (B) The morphological changes of cells treated with PTX or BEZ235 or both for 2 hours. Using β-tubulin staining (green fluorescence) and PI staining (red fluorescence) cell nuclei, observe the morphological changes from round cells in the control group to fibroblast-shaped cells in the treatment group. These images were taken with an Olympus IX51 fluorescence microscope at 400x magnification.

Colon cancer cells are prone to multidrug resistance. In order to study the mechanism of action of PTX and BEZ235 on HT29DDP and HCT116-LOHP colon cancer cells, we compared the expression of Pgp and multidrug resistance-related protein (ABCC1) between different treatment groups by Western blot analysis. Compared with the untreated control group, the expression of Pgp (Figure 4A-D) and ABCC1 (Figure 4E-H) was significantly reduced after single drug and NE-PTX treatment. As the dose of the drug increases, the decrease in protein expression becomes more significant. Although there was no significant difference in the expression of Pgp and ABCC1 protein in the BEZ235 50nM group, the combined treatment decreased more (P <0.05) (Figure 4A, B, E, F and Figure 4C, D, G, H for HT29DDP and HCT116- LOHP cells). The effect of PTX combined with BEZ235 and NE-PTX combined with BEZ235 on reducing the expression of resistance protein is more obvious than that of single drug application. Among them, the NE-PTX combined with BEZ235 group has the most obvious effect on reducing the expression of resistance protein. Figure 4 Expression of Pgp and ABCC1 in HT29DDP and HCT116-LOHP cells. Western Blotting (WB) test results showed that compared with untreated controls, Pgp (AD) and ABCC1 (EH) expressions were significantly reduced after single PTX and NE-PTX treatment; for HT29DDP and HCT116-LOHP cells, combined therapy The reduction in sigma is even more significant (P <0.05) (A, B, E, F and C, D, G, H). The graphs of b, d, and f. And h are the optical density measurement results of the corresponding WB measurement. Note: *P<0.05; **P<0.01.

Figure 4 Expression of Pgp and ABCC1 in HT29DDP and HCT116-LOHP cells. Western Blotting (WB) test results showed that compared with untreated controls, Pgp (AD) and ABCC1 (EH) expressions were significantly reduced after single PTX and NE-PTX treatment; for HT29DDP and HCT116-LOHP cells, combined therapy The reduction in sigma is even more significant (P <0.05) (A, B, E, F and C, D, G, H). The graphs of b, d, and f. And h are the optical density measurement results of the corresponding WB measurement.

Note: *P<0.05; **P<0.01.

We further analyzed the effect of combination therapy on the apoptosis of colon cancer cells. The results of HT29DDP and HCT116-LOHP cells (Figure 5A-D) respectively showed that the apoptosis rate of the different treatment groups was higher than that of the untreated control group; the difference between the PTX-16nM group and the control group was statistically significant (P<0.05). The difference between the combination treatment group (PTX-8nM+BEZ 50nM group and PTX-16nM+BEZ 50nM group) and the control group was also statistically significant (P<0.01). These data indicate that compared with PTX alone, PTX combined with BEZ235 can more effectively promote cell apoptosis in drug-resistant colon cancer. Then we further analyzed the level of Bcl-2 protein (an anti-apoptotic protein related to the mitochondrial apoptotic pathway) and found that treatment with a single drug resulted in a reduction of the protein, but a more significant reduction was seen in the combination ( Image 6). Compared with the untreated control, the expression of Bcl-2 was significantly reduced after treatment with paclitaxel and NE-PTX alone; for HT29DDP and HCT116-LOHP cells, the combined treatment even reduced it even more significantly (P <0.05). Among them, the NE-PTX combined with BEZ235 group showed the most obvious effect of reducing the expression of resistance protein, which was consistent with the above-mentioned cell death results. Figure 5 PI staining sub-G1/G0 population apoptosis analysis. After different treatments with PTX and BEZ235 in HT29DDP (A and B) and HCT116-LOHP (C and D) cancer cells, different degrees of apoptosis were observed. Figures a and c show the apoptotic sub-G1/G0 (P3) population. Figures c and d show the summary of the 3 individual determinations. Note: *P <0.05, **P <0.01. Figure 6 Apoptosis analysis of Bcl-2 (B-cell lymphoma 2) protein expression in HT29DDP and HCT116 LOHP cell lines. After PTX and BEZ235 were treated differently, the expression of Bcl-2 was significantly reduced after paclitaxel (PTX) and NE-PTX treatment alone; while the combined treatment reduced it more significantly (for HT29DDP (A and B) and HCT116-LOHP) Cells (C and D). Among them, the NE-PTX combined with BEZ235 group showed the most obvious effect of reducing this expression. Protein. Note: *P<0.05; **P<0.01.

Figure 5 Apoptosis analysis of sub-G1/G0 populations by PI staining. After different treatments with PTX and BEZ235 in HT29DDP (A and B) and HCT116-LOHP (C and D) cancer cells, different degrees of apoptosis were observed. Figures a and c show the apoptotic sub-G1/G0 (P3) population. Figures c and d show the summary of the 3 individual determinations.

Note: *P <0.05, **P <0.01.

Figure 6 Apoptosis analysis of Bcl-2 (B-cell lymphoma 2) protein expression in HT29DDP and HCT116 LOHP cell lines. After PTX and BEZ235 were treated differently, the expression of Bcl-2 was significantly reduced after paclitaxel (PTX) and NE-PTX treatment alone; while the combined treatment reduced it more significantly (for HT29DDP (A and B) and HCT116-LOHP) Cells (C and D). Among them, the NE-PTX combined with BEZ235 group showed the most obvious effect of reducing this expression. Protein.

Note: *P<0.05; **P<0.01.

To further verify the in vitro results, we used a xenograft tumor model in mice. HT29DDP and HCT116-LOHP cells were injected subcutaneously into nude mice. Mice in each group were photographed 3 weeks after tumor formation (Figure 7A). Mice with HT29DDP and HCT116-LOHP tumors gained weight, and the situation was similar between all animals, allowing modeling (Figure 7B). After the tumor reaches a predetermined volume, the drug is injected into the tumor. After 3 weeks, the tumor was dissected and weighed (Figure 7C). Compared with the untreated group, the tumor weight in the different treatment groups decreased, and the tumor weight in the combined treatment group decreased more significantly (Figure 7D) (P<0.005). This result confirms that the combination therapy can significantly inhibit tumor growth in vivo, which is consistent with the results obtained in vitro. Figure 7 Inhibition of tumor growth in nude mice after drug treatment. (A) Nude mouse tumor: (1) Control group, injected with PBS; (2) PTX, injected with 10mg/kg PTX; (3) BEZ235, injected with 20mg/kg BEZ235; (4) NE-PTX, injected with 6.6mg /kg; (5) NE-PTX + BEZ235, 20mg/kg injection. The mice were imaged on the 21st day after the injection of cancer cells. (B) The body weight of nude mice in different groups from day 0 to day 25. (C) (A) Images of anatomical tumors in each group. (D) Comparison of tumor weight between different groups. Note: *P <0.05; ** P <0.01.

Figure 7 Inhibition of tumor growth in nude mice after drug treatment. (A) Nude mouse tumor: (1) Control group, injected with PBS; (2) PTX, injected with 10mg/kg PTX; (3) BEZ235, injected with 20mg/kg BEZ235; (4) NE-PTX, injected with 6.6mg /kg; (5) NE-PTX + BEZ235, 20mg/kg injection. The mice were imaged on the 21st day after the injection of cancer cells. (B) The body weight of nude mice in different groups from day 0 to day 25. (C) (A) Images of anatomical tumors in each group. (D) Comparison of tumor weight between different groups.

Note: *P <0.05; ** P <0.01.

Our previous studies have shown that PTX and BEZ235 have a synergistic inhibitory effect on the growth of K-ras mutant or non-mutant colorectal cancer cells. The nanoemulsion loaded with the combination of PTX and BEZ235 showed further improved sensitivity and synergy. 21 However, these studies used common colon cancer cells. Nanoemulsion (NE) is a stable, transparent, low-viscosity, homogeneous, thermodynamically stable dispersion system formed by oil phase, water phase, surfactant and co-surfactant in appropriate proportions. Nanoemulsions can increase the solubility of poorly soluble drugs and improve the stability and bioavailability of drugs. 21-23,25 In this study, we aimed to explore whether the combination of NE-PTX and BEZ235 can treat colon cancer drug-resistant cell lines. The NE-PTX we prepared showed good stability in culture medium, PBS and pH5.8 buffer. The cell viability results show that the combination therapy can increase its inhibitory effect by reducing the IC50 of PTX and inhibit tumor growth in animal models. This indicates that the method has the potential to reduce the amount of PTX and its toxic and side effects, and proves its potential clinical application value in the treatment of colon cancer.

The cellular mechanisms of PTX acting on cancer cells (including those cells with K-ras gene mutations) include disturbing the dynamic balance between microtubules and tubulin dimers, preventing the cell cycle from entering the G2/M phase, and promoting apoptosis . 24,26,27 BEZ235 is a dual inhibitor of PI3K/Akt/mTOR signaling pathway, which can down-regulate the expression of ATP-binding transporters (such as Pgp), reverse Pgp-mediated multidrug resistance in colorectal cancer, and promote cell apoptosis . 20,28 Therefore, we hypothesized that PTX combined with BEZ235 can simultaneously act on the growth, proliferation, apoptosis and drug resistance of colon cancer cells, thereby achieving a synergistic anti-tumor and anti-drug resistance effect. The results of this study confirmed our hypothesis. In order to further improve the sensitivity of tumor cells to PTX, we used the previously reported nanoemulsion delivery (Hong Zou et al. 2016). We demonstrated that nanoemulsion delivery is more effective in reducing IC50 and inhibiting the growth of cancer cells in vitro and in vivo. In addition, it can be clearly seen that PTX+BEZ235 has a synergistic inhibitory effect on cancer cells, which is beneficial to a single drug. For future applications, nanoemulsions that load two drugs together may benefit this treatment method.

The mechanism of colon cancer drug resistance involves tumor cell resistance to apoptosis. It is understood that nearly half of colon cancer patients have K-ras gene mutations. Therefore, EGFR receptor blockers, such as cetuximab, become ineffective, which is also a major problem in clinical practice. PTX can effectively promote cell apoptosis, and tumor cells with K-ras mutations are also sensitive to PTX. 10,11,29 The main targets and functions of PTX include: (1) PTX is located in the tubulin/microtubule system. It promotes microtubule polymerization, inhibits microtubule degradation, and prevents cell division in the G2/M phase. (2) PTX causes abnormal autophagy transport and localization. In addition, it inhibits the degradation of autophagy, thereby inducing apoptosis and degradation. 10 BEZ235 can also inhibit the apoptosis of colon cancer cells. Therefore, we used flow cytometry to analyze the apoptosis of HT29DDP and HCT116-LOHP cells in different treatment groups of PTX and BEZ235. The results showed that the apoptosis rate of HT29DDP and HCT116-LOHP cells in the treatment group was higher than that in the negative control group. This suggests that PTX and BEZ235 can promote apoptosis in two colon cancer cell lines, albeit to different degrees. The apoptosis rate of the PTX-16nM group was significantly different from that of the control group (P <0.05), indicating that high concentrations of PTX induced a higher rate of apoptosis for both drug-resistant colon cancer cells. This indicates that PTX induces apoptosis in a concentration-dependent manner, which is consistent with the results of the CCK8 experiment. For example, the degree of apoptosis in the PTX-8nM+BEZ50nM group and PTX-16nM+BEZ50nM group was significantly higher than that of the control group (P <0.01). This further confirms that the combination of PTX and BEZ235 can more significantly promote the apoptosis of drug-resistant colon cancer cell lines. In addition, the combination therapy has a stronger inhibitory effect on tumor growth, can significantly promote cell apoptosis and inhibit tumor growth.

In MDR tumors, the ABC membrane transporter family helps tumor cells pump intracellular chemotherapeutic drugs out of the cell, which is an important mechanism for tumor cell resistance. 30 ABCB1/Pgp is the most important member of cells involved in MDR. Membrane transport function, and is the main target of tumor MDR inhibitors. 31 ABCC1/MRP1 and ABCB1/Pgp are energy-dependent drug efflux pumps with ATP binding sites and drug binding sites. ATP can be hydrolyzed to provide energy by combining with ABCC1 and Pgp. This energy is used to pump the drug into the cell, resulting in a decrease in the concentration of the drug in the cell or the formation of an interval distribution. Therefore, the intracellular drug concentration does not reach an effective level, leading to drug resistance and chemotherapy failure. Therefore, it is necessary to find new treatment methods for drug-resistant colon cancer. In this study, Western blotting was used to detect the protein expression of Pgp and ABCC 1 in the two cell lines. The results showed that after combined treatment with PTX and BEZ235, the expression of the two proteins was down-regulated compared with the untreated control, indicating that the combined treatment can reduce the multidrug resistance of the two drug-resistant colon cancer cell lines by inhibiting the protein levels of the two drug-resistant colon cancer cell lines. . porter. For example, the expressions of Pgp and ABCC1 in the PTX-16nM, BEZ50nM, and combination groups were lower than those in the PTX-8nM group, indicating that its inhibitory effect was better than that of the PTX-8nM group (Figure 3). In addition, the expressions of Pgp and ABCC1 in the PTX-8nM+BEZ50nM and PTX-16nM+BEZ50nM groups were lower than those in the BEZ50nM group, proving that the combined treatment has a stronger inhibitory effect than the single treatment of BEZ235. The expression of Pgp and ABCC1 in the PTX-16nM+BEZ50nM group was significantly lower than that in the PTX-16nM group (P <0.01), which further proved this point. Therefore, the combination of PTX and BEZ235 can inhibit or reduce the drug resistance of colon cancer by inhibiting the expression of ABCB1/Pgp. To verify the in vitro results, we tested the synergy of PTX and BEZ235 in nude mice. The results showed that the tumor weight of nude mice treated with PTX, BEZ235, NE-PTX, NE-PTX+BEZ235 was lower than that of untreated (control group) mice; the tumor weight of nude mice treated with NE-PTX and BEZ235 was the lowest. These results confirm the results of in vitro experiments. Compared with the PTX treatment group, the tumor weight in the NE-PTX and NE-PTX+BEZ235 treatment groups decreased more significantly. In conclusion, our data shows that PTX loaded in nanoemulsion and combined with BEZ235 can effectively overcome drug resistance and increase the drug sensitivity of drug-resistant colon cancer cells, which is expected to become a potential method for the treatment of drug-resistant colon cancer.

PTX combined with BEZ235 can effectively inhibit the proliferation of drug-resistant colon cancer HT29DDP and HCT116-LOHP cells in vitro and in vivo. This mechanism is mainly related to increasing cell apoptosis and inhibiting multidrug resistance. Combined with previous studies, 21 showed that the combination of PTX and low concentrations of BEZ235 is a promising method not only for the treatment of ordinary colon cancer cells, but also for the treatment of platinum-resistant colon cancer cells. The use of nanoemulsion delivery system further increases the sensitivity of tumor cells to PTX. NE-loaded PTX has the characteristics of good stability and increased solubility. Therefore, the clinical application of this method should be further explored in the treatment of colon cancer.

All authors have made significant contributions to the concept and design, data acquisition or data analysis and interpretation; participated in drafting articles or critically revised important knowledge content; agreed to submit to the current journal; finally approved the version to be published; and agreed Responsible for all aspects of work.

This research was funded by the National Natural Science Foundation of China [Grant No. 81660411, 81460383], China Xinjiang Production and Construction Corps International Cooperation Project [Grant No. 2019BC001], Xinjiang Production and Construction Corps Key Field Innovation Team Project, China [Grant No. 2018CB002].

Yali Hu, Kunpeng Zhang, and Xingyao Zhu are the co-first authors of this study. The author declares that there is no conflict of interest.

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