Low temperature upregulating HSP70 expression to mitigate the paclitaxel-induced damages in NHEK cell

Abstract

Scalp cooling is the most approved treatment for preventing chemotherapy-induced alopecia (CIA). However, the protective mechanism of scalp cooling has rarely been reported. The goal of the present study was to study the relationship between paclitaxel concentration and temperature and the inhibitory effect of low temperature on paclitaxel-induced alopecia. The results showed that the dose of paclitaxel should not exceed 60-70 mg/mL during scalp cooling treatment, and the optimal cooling temperature under different paclitaxel concentrations was determined. Normal human epidermal keratinocytes (NHEK) cells were analyzed by global transcriptome analysis, functional annotation and pathway analysis of differentially expressed genes (DEGs) and ELISA kit to analyze the mechanism of low temperature therapy. The expression of HSPA8, HSPA1A and HSPA1B, which belongs to HSP70, was up-regulated by low temperature. These genes are important target genes of low temperature treatment, which were confirmed by ELISA. The up-regulation of PLK2 and the down-regulation of TXNIP expression are the upstream of mitochondrial dysfunction and ROS, inhibiting the accumulation of ROS and up-regulating the mitochondrial membrane potential. Our research partially elucidates the therapeutic mechanism of scalp cooling, which provides a new idea on the drug research and development in CIA.

Keywords: Differentially expressed genes; HSP70; Optimal cooling temperature; Scalp cooling.

Figure 1. Effect of paclitaxel concentration and temperature on cell survival.

(A) After paclitaxel treatment for 2 h, CCK-8 was used to measure cell survival rate. There were three biological replicates for each group. The lower the paclitaxel concentration and the lower the temperature, the higher the cell survival rate (n = 3). Data are the mean ± s.e.m. One-way analysis of variance (ANOVA). (B) NHEK cells were treated with different concentrations of paclitaxel and different temperatures. The iso-activity line ranges from a high concentration of taxol at low temperatures to a low concentration of taxol at high temperatures. As the temperature increases, the iso-activity curve becomes S-shaped.

Figure 2. Differentially transcribed genes following low temperature stress treatment of NHEK cells. The same batch of cells were divided into two groups, one was the control group and the other was the 22 °C treatment group. Three biological replicates were used.

(A) The DEG volcano plot showed all the sampled genes. Red points represented the up-regulated genes; green points represented the down-regulated genes; grey points represented the not DTGs. (B) Gene ontology (GO) enrichment analysis of DEG. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function. (C) Go enrichment analysis for DEGs. (D) The KEGG pathway enriched analysis significantly (FDR < 0.05) for the DEGs. Rich factor refers to the ratio of the number of differentially enriched genes to the number of annotated genes in the pathway. The rich factor represents the degree of enrichment. (E) Cluster analysis of DEGs. Red and blue indicated higher and lower expression genes, respectively.

Figure 3. Expression pf HSP70, MMP and ROS of NHEK cells.

(A) The concentration of HSP70 was tested using the HSP70 ELISA kit. The concentration of HSP70 increased significantly at 22 °C and firstly increased and then decreased with the increase of exposure time (n = 3). (B) The 37 °C and 22 °C groups were all treated with paclitaxel for 2 h. The MMP increased significantly after paclitaxel treatment, the MMP decreased significantly after low temperature treatment (n = 3). CCCP group is a positive control group. (C) The ROS of cells significantly decreased after low temperature treatment (n = 3). There were three biological replicates for each group. Data are the mean ±s.e.m. One-way analysis of variance (ANOVA). * P < 0.05, ^∗∗P < 0.01.

Figure 4. The cell survival rate and expression of HSP70 of NHEK cells.

(A) The 10,000 cells were treated with 12.5 µM HSP70 agonist TRC051384 and 100 µM HSP70 inhibitor KNK437 for 2 h (n = 3). (B) The 10,000 cells were treated with 12.5 µM HSP70 agonist TRC051384 and 100 μM HSP70 inhibitor KNK437 for 2 h (n = 3). The concentration of HSP70 was tested by HSP70 ELISA kit. There were three biological replicates for each group. Data are the mean ± s.e.m. One-way analysis of variance (ANOVA). **P < 0.01.

Figure 5. Relationship between scalp temperature and cell survival under different paclitaxel concentrations.

The critical treatment temperature under different paclitaxel doses can be calculated.

Figure 6. Schematic diagram of paclitaxel damage reduction mechanism by low temperature.

When the cells were stimulated by low temperature at 22 °C, the mRNA changed. Red is ROS related mRNA and blue is apoptosis related mRNA.