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Abstract

Cryptotanshinone (CPT) is the main pharmacologically active component of Salvia miltiorrhiza Bunge, and has been demonstrated to have inhibitory effects on a variety of cancer cell types. However, the target(s) of CPT in colorectal cancer (CRC) cells are still to be identified. The aim of this study was to investigate the molecular mechanism of CPT-induced apoptosis in CRC cells. Transcriptome sequencing revealed that 1234 genes were differentially expressed in CPT-treated SW480 cells compared with vehicle control, which were associated with signaling pathways such as PI3K/AKT, Pathways in cancer and biological processes include cell proliferation and mitochondrial function. CCK-8, colony formation assay, and CRC tumor xenograft models suggested that CPT suppressed the colony formation ability and inhibited proliferation of CRC cells in vitro and in vivo, while flow cytometry indicated that CPT arrested the cell cycle in S phase. JC-1, Hoechst and Western blot analysis indicated that CPT promoted apoptosis in CRC cells and triggered alterations in mitochondrial membrane potential. In addition, database analysis showed that high SIRT3 expression in CRC and high SIRT3 expression in CRC was associated with shorter survival times. Immunofluorescence, Western blot, and molecular docking confirmed that CPT was able to target SIRT3 and downregulate SIRT3 protein levels in a concentration-dependent manner. In summary, CPT inhibits SIRT3 protein expression and inhibits CRC proliferation in vitro and in vivo.

Keywords

cryptotanshinone colorectal cancer proliferation apoptosis mitochondria SIRT3

Introduction

Colorectal cancer (CRC) is the third most common malignant tumor in the world, with an increasing incidence and deaths year-on-year.¹ A combination of chemotherapy and radiotherapy, as well as biological targeting therapies, are commonly used in clinical CRC treatment to improve the quality and duration of patients survival.² Moreover, drugs with lower toxicity and better targeting effects for chemotherapy or that assist radiotherapy are being sought and developed to enhance treatment efficacy and safety.

The dried root and rhizome of Salvia miltiorrhiza Bunge (Danshen in Chinese), family Labiatae, is a traditional Chinese medicine.³ Cryptotanshinone (CPT) is one of the main tanshinone compounds (accounting for about 30% of the total tanshinones), with anti-tumor, anti-inflammatory, cardiovascular protection, anti-fibrosis, and anti-metabolic disorder properties.^4–7 CPT can inhibit the proliferation of a variety of cancer cells, such as lung cancer, gastric cancer, liver cancer, and bladder cancer, as well as CRC.⁸ It is reported that CPT intervenes in the progression of CRC, including proliferation and angiogenesis.⁹ In CRC cells (SW620/AD300), CPT overcame P-glycoprotein (P-gp)-mediated multidrug resistance by decreasing P-gp mRNA and protein expression, which in turn inhibited P-gp ATPase activity, resulting in increased toxicity of doxorubicin to SW620/AD300 cells.¹⁰ Moreover, CPT induces reactive oxygen species (ROS)-dependent autophagy in CRC cells by increasing p38 mitogen-activated protein kinase (MAPK) phosphorylation levels.¹¹ CPT induces the apoptosis of HCT116 and SW480 cells by inhibiting signal transducer and activator of transcription 3 (STAT3) phosphorylation and down-regulates the expression of its downstream targets CyclinD1, Survivin and Bcl-2, which further inhibit cell proliferation.¹² In addition, CPT induces p53-independent apoptosis and autophagy in HCT116 CRC cells.¹³

Deacetylase sirtuin-3 (SIRT3), a NAD + -dependent class III histone deacetylase, belongs to the Sirtuins family.¹⁴ SIRT3, mainly localized at mitochondria, harvests numerous substrates such as hypoxia-inducible factor 1-alpha (HIF-1a) and tumor suppressor p53 (p53), which are further involved in the development and progression of cancer and neurological diseases, as well as cardiovascular diseases.¹⁵ Tumor tissues often present aberrant SIRT3 and acetylation levels.¹⁶ SIRT3 is highly expressed in CRC, and, therefore, a series of small molecule SIRT3 inhibitors have been investigated for CRC treatment.¹⁷ It is reported that there is a positive correlation between SHMT2 acetylation and SIRT3 expression in 309 CRC tumor samples, and patients with high SIRT3 expression had shorter survival.¹⁸ SIRT3 knockdown was able to significantly inhibit H1299 cell growth and proliferation and induce apoptosis.¹⁹ In our study, we explored the potential targets of CPT-CRC by transcriptome analysis. Further we demonstrated that CPT could suppress CRC cell (SW480 and SW620) proliferation in vivo and in vitro, driving mitochondria dependent apoptosis, and inhibition of SIRT3 expression.

Results

Transcriptome Sequencing

To elucidate the mechanism of CPT in regulating CRC, we performed transcriptome sequencing. The chemical structure is shown in Figure 1A (PubChem CID: 160254), and with CPT treatment, 1234 differentially expressed genes were identified in SW480 cells. In detail, 679 genes were upregulated and 555 downregulated (fold change cutoff of ≥ 1.3 and adjusted FDR ≤ 0.001) (Figure 1B). Kyoto Encyclopedia of Genes and Genomes (KEGG) (Q value ≤ 0.05) enrichment analysis against the differential genes suggested that CPT regulates several signaling pathway that are tightly related with CRC proliferation and metastasis, including the MAPK signaling pathway, proteoglycans in cancer, focal adhesion, and cancer-related signaling pathways (Figure 1C). Correspondingly, Gene Ontology (GO) analysis demonstrates that CPT alters CRC apoptosis, positive regulation of epithelial to mesenchymal transition, angiogenesis, cellular senescence, mitochondrial respiratory chain complex I assembly, and cell migration of biological processes, which is mediated by related cell composition and molecular function (Figure 1D). These data suggest that CPT exerts its activity against CRC by regulating multiple targets and their associated biological processes and signaling pathways.

Figure 1.

Transcriptome sequencing. (A) CPT structure. (B) Differential gene volcano plot. Red, green, and gray indicate upregulated genes, downregulated genes, and genes that are not differentially expressed, respectively. (C) Top 25 signaling pathways in CRC treated with CPT (Q value ≤ 0.05). Larger and redder dots indicate greater enrichment of genes in this pathway with smaller P values. (D) Top 20 enriched terms after CPT treatment, including cell composition (CC), biological process (BP), and molecular function (MF) terms.

CPT Inhibits the Proliferation of SW480 and SW620 in vitro and in vivo

SW480 and SW620 cells were treated with different concentrations of CPT (0, 0.5, 1, 2, 4, 8, 16, and 32 μM) for 48 h, CCK-8 experiment showed that CPT inhibited the viability of SW480 and SW620 in a dose-dependent manner (Figure 2A). Thus, we selected 0.5, 1, and 2 μM concentrations of CPT for subsequent experiments. These CPT concentrations not only significantly inhibited the cell viability, but also suppressed the ability of colony formation of SW480 and SW620 cells (Figure 2B and C). Consistent with this, flow cytometry analysis clarified that CPT arrest of SW480 and SW620 cells was in the S phase, resulting in a decreased percentage of G1 and G2/M phase cells compared to the control group (Figure 2D and E).

Figure 2.

CPT inhibits SW480 and SW620 cell proliferation. (A) CCK-8 assay of CRC cell proliferation after treatment with CPT. (B, C) Colony formation assays and corresponding quantification. (D) Cell cycle detection by flow cytometry. (E) Graph of cell cycle quantification. As determined by 1-way ANOVA analysis of the data, *P ˂ .05, **P ˂ .01, ***P ˂ .001, and ****P ˂ .0001 means significant difference compared to the control group.

To evaluate the pharmacological activity of CPT in the treatment of CRC in vivo, a xenograft of CRC tumor models in nude mice was constructed. Compared to the control group, CPT (20 mg/kg/d) treatment significantly reduced tumor volume (Figure 3A and B). Furthermore, the difference in body weight between the control and CPT-treated groups was also estimated. The body weight of the SW480 cell xenograft group was not statistically significantly different, whereas the SW480 cell xenograft group exhibited a significant increase in body weight with CPT treatment (Figure 3C), which indicated a low toxicity of CPT. Together, these results indicated that CPT inhibits SW480 and SW620 proliferation in vivo and in vitro.

Figure 3.

CPT inhibits tumor growth in vivo. (A) Tumor volume after CPT treatment. (B) Tumor volume curves. (C) Changes in body weight of mice administered CPT. *P ˂ .05, **P ˂ .01 indicate a significant difference from corresponding control group.

CPT Induces CRC Cell Apoptosis and Triggers Alteration of Mitochondrial Membrane Potential

Flow cytometry analysis showed that CPT induced apoptosis of SW480 and SW620 cells (Figure 4A and B, Supplemental Figure 1A and B). Consistent with this, Hoechst staining showed an increase of densely stained nuclei and apoptotic bodies with increasing CPT concentrations (Figure 4C and D, Supplemental Figure 1C and D), which demonstrated that CPT drives CRC cell (SW480 and SW620) apoptosis. Moreover, JC-1 staining showed that CPT treatment significantly decreased the red fluorescence while inducing the green fluorescence of SW480 and SW620, resulting in a decreased mitochondrial membrane potential, which is a characteristic of apoptosis (Figure 4E, Supplemental Figure 1E). Moreover, apoptosis related proteins were detected. As shown in Figure 4F and G and Supplemental Figure F and G, the expression of Bax and Cytc was increased by CPT in a dose-dependent manner, while Bcl2 and Caspase 3 expression was decreased, which confirmed that CPT can induce apoptosis in CRC cells and trigger the alteration in mitochondrial membrane potential.

Figure 4.

CPT induces SW480 cell apoptosis and triggers changes in mitochondrial membrane potential. (A, B) Flow cytometry quantitative analysis of the apoptosis rate of SW480 cells and quantitative analysis. (C) Hoechst staining of cell morphological changes. Blue fluorescence indicates staining of nuclei or apoptotic bodies. Magnification × 10. (D) Quantitative analysis of Hoechst staining. (E) JC-1 detects changes in mitochondrial membrane potential. The red fluorescence is produced by JC-1 forming a polymer in the mitochondrial matrix, and the green fluorescence is produced by the JC-1 monomer. The ratio of green to red fluorescence reports on the mitochondrial membrane potential. Magnification × 10. (F, G) Western blot analysis of the protein expression of Bax, Bcl2, Caspase 3, and Cytc (F) and corresponding quantification (G). *P ˂ .05, **P ˂ .01, ***P ˂ .001, and ****P ˂ .0001.

SIRT3 Overexpression is Correlated With CRC Patient Survival

Based on the publicly available omics data, we analyzed the relationship between CRC and SIRT3 expression. Analysis revealed that the expression level of SIRT3 was different in various cancer types (Figure 5A). Analysis of clinical cases in the cancer genome atlas (TCGA) database (https://portal.gdc.cancer.gov/; 41 normal samples and 286 CRC samples) indicated that SIRT3 was overexpressed in CRC samples (Figure 5B), which correlated with patient survival (Figure 5C). This suggests that SIRT3 is associated with CRC development, and could be a potential target in CRC treatment.

Figure 5.

SIRT3 overexpression is correlated with survival in CRC. (A) SIRT3 expression in different cancer cells. (B) SIRT3 expression in TCGA clinical samples (common sample, n = 41 and tumor sample, n = 286). (C) Expression of SIRT3 in CRC and survival analysis. Blue indicates low expression of SIRT3, red indicates high expression of SIRT3.

CPT Targets SIRT3 and Downregulates SIRT3 Protein Expression

To explore further the relationship between CPT and SIRT3, we performed immunofluorescence, Western blot analysis, and molecular docking experiments. Immunofluorescence (Figure 6A) and Western blot analysis (Figure 6B and C) demonstrated that with CPT treatment, the SIRT3 protein level was down-regulated both in SW480 and SW620 cells in a dose-dependent manner. On the other hand, the molecular docking experiment indicated that CPT could bind with Ile154, Pro155, Phe157, Arg158, Phe180, Leu199, Asn229, and Ile230, as well as His248 of SIRT3. The binding energy was −8.6 kcal/mol (<−5 kcal/mol), which indicated that the interaction of CPT and SITR3 is stable (Figure 6D). Thus, these findings indicated that CPT targets SIRT3 and downregulates SIRT3 protein levels.

Figure 6.

CPT targets SIRT3 and downregulates SIRT3 protein expression in SW480 and SW620 cells. (A) Immunofluorescence was used to detect the effect of CPT on SIRT3 expression. Magnification × 20. (B, C) Western blot were used to analyze the protein expression of SIRT3 (B) and corresponding quantification (C). (D) Molecular docking. *P ˂ .05 and ***P ˂ .001 indicate a significant difference from control.

Discussion

In this study, we explored the potential mechanism of CPT in the treatment of CRC through transcriptome sequencing, which revealed that CPT regulated CRC procession through apoptosis, angiogenesis, and EMT. Further experiments demonstrated that CPT suppresses CRC proliferation in vitro and in vivo, inhibits the colony formation ability of CRC cells, and drives CRC apoptosis. Moreover, CPT targets and inhibits the expression of SIRT3, which is tightly related to CRC progression and CRC patient survival. This indicates a potential role of CPT as a SIRT3 inhibitor in CRC treatment.

Several signaling pathways are critical in CRC progression, such as the MAPK and PI3K/AKT signaling pathways. It is reported that Mex3a promotes carcinogenesis in CRC by activating the RAP1/MAPK signaling pathway.²⁰ While inhibition of MAPK signaling was able to inhibit CRC cell proliferation,²¹ on the other hand, the PI3K/AKT signaling pathway is critical in CRC cell autophagy and epithelial-mesenchymal transition.²² Consistent with this, KEGG analysis based on transcriptome sequencing data, identified the PI3K/AKT and MAPK signaling pathways in the CPT-CRC signaling pathways (Figure 1). This relationship was confirmed by Zhang et al, who demonstrated that CPT inhibits CRC cell proliferation by reducing the phosphorylation of PI3K, AKT, and MTOR,⁹ indicating that CPT can treat CRC by mediating the indicated signaling pathways. Recently, CPT was identified as a compound with novel anti-CRC pharmacological activity. It is reported that CPT increased the susceptibility of CRC cells to endoplasmic reticulum stress-mediated apoptosis by reducing calpain activity.²³ Our study showed that 0.5, 1, and 2 μM CPT were able to inhibit CRC cell proliferation both in vivo and in vitro and suppress the colony formation ability of SW480 and SW620 (Figures 2 and 3). However, a prior study found that CPT dramatically reduced SW620 and HCT116 proliferation in a dose- and time-dependent manner (48 h, 10 μM), although SW480 cells showed no significant cytotoxic effect.²⁴ Additionally, CPT can prevent STAT3 phosphorylation at Tyr705, which in turn drives the apoptosis of hepatocellular carcinoma cells and tongue squamous cell carcinoma cells, as well as inhibiting gastric cancer cell growth.^25–27 Relevant studies have found that p-STAT3 levels are elevated in CRC,²⁸ while blocking STAT3 signaling significantly reduced CRC cell viability.²⁹ Subsequently, it was demonstrated that STAT3 enhances the aggressiveness of CRC cells by increasing the expression of matrix metalloproteinases (MMP-1, MMP-3, MMP-7, and MMP-9).³⁰ Therefore, we postulate that CPT may also, through preventing STAT3 activity, affect the onset and spread of CRC; this hypothesis can be verified by future research.

Mitochondria are key metabolic organelles that play a central role in regulating apoptosis.³¹ It has been shown that acriflavine impairs mitochondrial function through upregulation of P53 mRNA and protein, which in turn increases chemosensitivity in SW620 cells.³² In the present study, CPT induced CRC apoptosis and triggered alterations in mitochondrial membrane potential (Figure 4), suggesting that CPT may induce mitochondrial apoptosis by altering mitochondrial metabolism and thereby exerting anti-CRC effects.

The expression level of SIRT3 is different in various cancer tissues, indicating that SIRT3 plays the role of oncogene or tumor suppressor, mediates substrates acetylation, and regulates the indicated signaling pathway. SIRT3 is highly expressed in CRC, oral cancer, lung cancer, melanoma, and renal cancer.^33,34 In CRC cells, downregulation of SIRT3 led to a decrease in PTEN expression, which in turn triggered mitochondrial fission to induce apoptosis.³⁵ In addition, small molecules or external conditions can affect the development and progression of CRC by targeting SIRT3. For instance, δ-valerobetaine induces apoptosis in CRC cells by downregulating the expression of SIRT3.³⁶ In addition, cisplatin induced mitochondrial methylenetetrahydrofolate dehydrogenase (MTHFD2) acetylation by down-regulating the expression of SIRT3 and inhibiting SIRT3 deacetylation, resulting in decreased NADPH levels and increased ROS content, which in turn reduced CRC viability.³⁷ These investigations compensate for our flaws. In our study, we identified that CPT down-regulates the protein level, targets Ile154, Pro155, Phe157, Arg158, Phe180, Leu199, Asn229, and Ile230, as well as His248 residues of SIRT3, further functions in inhibiting the proliferation and inducing apoptosis of CRC (Figure 7). On the other hand, SIRT3 has been identified as a tumor suppressor in ovarian,³⁸ breast,³⁹ and liver cancers.⁴⁰ According to related research, CPT inhibits the proliferation of ovarian cancer cells by down-regulating STAT3, further inducing SIRT3 expression.⁴¹ Whether the regulation of CPT on SIRT3 protein level is STAT3 mediated or direct needs further investigation. However, SIRT3 harvests several substrates, such as KU70. It is reported that SIRT3 mediates the de-acetylation of KU70 and BAX, further inhibiting BAX related apoptosis. We speculate whether CPT could regulate the acetylation of KU70 and interrupt the interaction of KU70 and BAX, further regulating CRC cell apoptosis.^42,43 Related experiments will be performed in the future.

Figure 7.

CPT prevents the growth of CRC cells through molecular processes. CPT targets and inhibits protein levels of SIRT3, triggers mitochondrial membrane potential alteration, and drives CRC cell apoptosis, which in turn inhibits CRC cell proliferation in vivo and in vitro.

Conclusions

In summary, we suggest that CPT inhibits CRC cell proliferation in vivo and in vitro, drives CRC cell apoptosis, and targets and suppresses the protein level of SIRT3, thus providing novel ideas for CPT treatment of CRC. CPT-CRC is associated with signaling pathways such as the PI3K-Akt signaling pathway, pathways in cancer, and biological processes such as cell proliferation and mitochondrial function. These findings establish a pathway to develop a potential SIRT3 inhibitor as an activator of the apoptotic pathway for CRC treatment.

Materials and Methods

Materials and Reagents

CPT was purchased from Shanghai Yuanye Biotechnology Co., Ltd (99% purity; B21586; Shanghai, China). Anti-B cell lymphoma 2 (Bcl2; 4223S), anti-Bax (2772), anti-cytochrome c (Cytc; 136F3), and anti-Caspase 3 (9662) antibodies were obtained from Cell Signaling Technology (CST; Massachusetts, USA). Anti-GAPDH (10494-1-AP), anti-SIRT3 (10099-1-AP), goat anti-mouse IgG HRP (SA00001-1), and goat anti-Rabbit IgG HRP (SA00001-2) antibodies were obtained from Proteintech (Wuhan, China). The Annexin V-FITC/PI Apoptosis Detection Kit (556547) was purchased from Becton, Dickinson and Company (BD; New Jersey, USA). Hoechst 33342 staining solution (C0031), dimethyl sulfoxide (DMSO; D8370), Triton X-100 (T8200), bovine serum albumin (BSA; A8850), and mitochondrial membrane potential kit (JC-1; M8650) were purchased from Solarbio (Beijing, China). Dulbecco's modified Eagle's medium (DMEM; C11995500BT) and fetal bovine serum (FBS; 16140071) were obtained from Gibco (California, USA). Phosphate buffered saline (PBS; SH30256), trypsin (SH30042), and penicillin–streptomycin dual antibody (SV30010) were from Hyclone (Logan, UT, USA). Cell counting kit-8 kit (CCK-8; AR1199) was purchased from BOSTER Biological Technology Co. Ltd (Wuhan, China). Propidium iodide (PI; CF0031) was obtained from Beijing Dingguo Changsheng Biotechnology (Beijing, China), Paraformaldehyde (BL539A) from Biosharp (Hefei, China), the chemiluminescence reagent (BeyoECL Moon) (P0018FS), and 4’, 6-diamino-2-phenylindole (DAPI) staining solution (C1005) from Biyuntian Biotechnology Co., Ltd (Shanghai, China), Trizol (9108) and TB Green (RR820A) from TaKaRa (Kusatsu, Shiga Japan), and the reverse transcription kit (K1622) from ThermoFisher Scientific (Massachusetts, USA). All other chemicals were obtained from commercial sources.

Animal Experiment

Male BALB/c-nu nude mice (4-6 weeks, weight 20 ± 2 g) were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd (Changchun, China). The experiment was carried out in the barrier facility of the Laboratory Animal Center of Jilin University and approved by the Laboratory Animal Welfare Ethics Committee of Jilin University (SY20191210). Animal feeding and protocols used were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 8023, revised 1978). Nude mice were randomly divided into 4 groups: control-SW480 (n = 11), CPT-SW480 (n = 11), control-SW620 (n = 8), and CPT-SW620 (n = 8). Either SW480 or SW620 cells (5 × 10⁶) were subcutaneously injected into the lateral ribs of nude mice to establish CRC mouse models. After 10 days of culture in the specific pathogen free environment, the control group received normal saline and the CPT group received CPT (20 mg/kg/day) by gavage for 14 days. During treatment, mice were weighed regularly (on days 2, 4, 6, 8, 10, 12, 14), and tumor size was measured with calipers on days 1, 3, 5, 7, 9, 10, 11, and 13.

Tumor volume was calculated using the following formula: tumor volume = 0.5 × L × W², where L and W are the maximum and minimum diameter, respectively.

Cell Culture

Human CRC cells SW480 and SW620 were purchased from Wuhan Punosai Life Technology Co., Ltd (Wuhan, China). Cultures were maintained at 37 °C in a humidified atmosphere using DMEM complete medium (10% FBS, 1% penicillin and streptomycin).

Transcriptome Sequencing

RNA Preparation

SW480 cells were seeded in 10 cm cell culture dishes. SW480 was maintained in DMEM medium containing 10% FBS for 24 h prior to addition of CPT until the cells adhered. CPT (5 μM) was added for 48 h, and cells were collected. Trizol lysis buffer was added to the cell sample tube (1 mL Trizol for 1 × 10⁶ cells). The supernatant was transferred to a centrifuge tube with 300 µL chloroform/isoamyl alcohol (24:1), mixed thoroughly by shaking and centrifuged at 12000 × g for 8 min at 4 °C. The supernatant was transferred to a 1.5 mL centrifuge tube, and 2/3 volume of isopropyl alcohol was added. The tube was gently inverted to mix, and placed at −20 °C for at least 2 h. The samples were then centrifuged at 17500 × g for 25 min at 4 °C. The supernatant was discarded, and the pellet was washed with 0.9 mL 75% ethanol, then centrifuged at 17500 × g for 3 min at 4 °C. The supernatant was discarded and the samples were centrifuged briefly following which residual liquid was removed and the samples were air dried for 3 to 5 min. The resulting pellet was resuspended in 20 to 200 μL DEPC-H₂O or RNase-free water.

mRNA Library Preparation

Secondary structures were denatured by heating, and oligo dT magnetic beads were used to enrich mRNA. Cleavage reagent was added and reacted until the mRNA was fragmented. Then a synthesis reaction system was added to prepare cDNA and add A bases to the 3’ end to ligate adapters to the cDNA. A PCR reaction system was prepared and the reaction program was set to amplify the ligation product. After the PCR product was denatured into a single strand, a circularization reaction system was prepared to obtain a single-stranded circular product, and the final library was obtained by digesting the uncircularized linear DNA molecules. Fragment size and library concentration were determined using an Agilent 2100 Bioanalyzer. The DNA molecules were sequenced using combined probe-anchored polymerization (cPAS) to obtain sequencing reads of 50 bp/100 bp/150 bp.

RNAdenovo Analysis Method

The raw data were filtered with SOAPnuke (v1.4.0) by (1) Removing reads containing adapters (adapter contamination); (2) Removing reads whose unknown base (“N” base) ratio is more than 5%; (3) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; afterwards clean reads were obtained and stored in FASTQ format. Trinity (v2.0.6) was used to assemble the clean reads and BUSCO to assess the assembly quality. Annotating assembled Unigene with 7 major functional databases (KEGG, GO, NR, NT, SwissProt, Pfam, and KOG), and transcription factors were predicted. PossionDis was performed between-group differential gene analysis with a fold change cutoff of ≥ 1.3 and FDR ≤ 0.001. The pheatmap function was used on the differential gene set to draw a heatmap of differential gene clusters.

Functional Enrichment

According to the GO and KEGG annotation results and official classifications, the differentially expressed genes were functionally classified, the phyper function in R software was used for KEGG enrichment analysis, and the TermFinder package was used for GO enrichment analysis (https://metacpan.org/pod/GO::TermFinder). With a Q value of ≤ 0.05 as the threshold, genes that met this condition were defined as being significantly enriched.

CCK-8

To determine the effect of CPT on the viability and proliferation of CRC cells, SW480 and SW620 were cultured in 96-well plates at a density of 5000 cells/well for 24 h. SW480 and SW620 cells were treated with CPT dissolved in DMSO at different concentrations (final concentrations of 0.5, 1, 2, 4, 8, 16, and 32 μM) for 48 h. The control group was treated with DMSO. Subsequently, 20 μL CCK-8 solution was added to each well for live cell staining, and incubated at 37 °C for 30 min, protected from light. Then the absorbance of each well at 450 nm was measured.

Colony Formation

SW480 and SW620 cells were seeded in 6-well plates at a density of 500 to 1000 cells/well. CPT (0, 0.5, 1, and 2 μM) was added to the cells after attachment, and fresh complete medium was replaced 48 h later; the medium was changed every 2 days. Two weeks later, they were stained with crystal violet and analyzed using Image J calculation (National Institutes of Health; Bethesda, Maryland, USA).

Cell Cycle

SW480 and SW620 cells in logarithmic growth phase were inoculated into 6-well plates at 2.5 × 10⁵/well for 24 h and treated with CPT for 48 h. Cells were collected with EDTA-free trypsin and fixed overnight in 70% ethanol. Cells were centrifuged for 5 min and stained with PI for 30 min in the dark. Cell cycle changes were detected using a Beckman Coulter flow cytometer (Beckman CytoFLEX; Brea, CA, USA). Cell cycle curves were plotted with GraphPad Prism 7 software (GraphPad Software; San Diego, CA, USA).

Apoptosis

SW480 and SW620 cells were seeded at a density of 2.5 × 10⁵ cells/well and treated with CPT for 48 h. Cells were collected into 1.5-mL Eppendorf tubes with trypsin, washed twice with PBS. Then the Annexin V-FITC/PI Apoptosis Detection Kit (binding buffer, FITC, propidium iodide) was used to complete the relevant operations. The apoptosis rate was detected by flow cytometric (Beckman CytoFLEX; Brea, CA, USA) analysis. SW480 and SW620 cells treated with CPT were then incubated with Hoechst staining solution for 30 min. After washing with PBS, the cell morphology was observed under a fluorescence microscope (Olympus; Tokyo, Japan). SW480 and SW620 cells treated with CPT were also incubated with JC-1 staining working solution for 20 min in the dark and then washed twice with 1× JC-1 buffer. After adding fresh medium, cells were observed under a fluorescence microscope (Olympus; Tokyo, Japan).

Western Blot

SW480 and SW620 cells in logarithmic growth phase were seeded in 6-well plates. Each well contained 1.5 × 10⁵ cells. The first well served as the vehicle control (DMSO) and CPT was added at different concentrations to the remaining wells. After 48 h incubation, cells were collected using trypsin, washed 2 to 3 times with PBS, lysed directly by adding an appropriate amount of 2 × loading buffer, sonicated at 20 Hz for 60 s, and boiled at 95 °C for 15 min. These samples were run on 10% SDS-PAGE gels, and after completion of electrophoresis and transfer, 5% skimmed milk was added and incubated at room temperature for 90 min. Primary antibodies against Bax, Bcl2, Cytc, Caspase 3, and SIRT3 were added and incubated at 4 °C overnight. The membranes were incubated with secondary antibodies for 90 min at room temperature and developed using an ECL kit.

Immunofluorescence

SW480 and SW620 cells were seeded in 6-well plates (1.5 × 10⁵, 2 × 10⁵ cells/well) containing DMEM complete medium for 24 h, and then the cells were exposed to CPT (0.5, 1 μM) for 48 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100 for 5 min, and nonspecific protein sites were blocked with 1% BSA for 1 h. Following addition of anti-SIRT3 (1:300) primary antibody, cells were incubated for 1 h at 37 °C, rinsed 3 times with PBS, and incubated with goat anti-mouse IgG/fluorescein isothiocyanate (1:500) for 1 h at 37 °C in the dark. Subsequently, nuclei were labeled using DAPI and images were acquired using confocal fluorescence microscopy (Olympus; Tokyo, Japan).

Molecular Docking

SDF structure files of CPT were obtained from the pubchem website, which further converted into PDB files using OpenBabel 2.3.2 software. The SIRT3 (PDB ID: 4O8Z) structure was obtained from the protein databank. AutoDockVina1.1.2. was used for molecular docking.

Statistical Analysis

All data are presented as the mean ± standard deviation (SD) of 3 independent experiments, and statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software; San Diego, CA, USA). Student's t-test was used for comparisons between 2 groups, and 1-way ANOVA was used to assess mean differences between 3 or more groups. A P-value less than .05 was considered to be statistically significant, *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Supplemental Material

sj-jpg-1-npx-10.1177_1934578X231194808 - Supplemental material for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo

Supplemental material, sj-jpg-1-npx-10.1177_1934578X231194808 for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo by Dimeng Song, Xiaoxue Fang, Zhiyuan Sun, Zhidong Qiu, Da Liu, Haoming Luo, Ye Jin and Donglu Wu in Natural Product Communications

Supplemental Material

sj-docx-2-npx-10.1177_1934578X231194808 - Supplemental material for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo

Supplemental material, sj-docx-2-npx-10.1177_1934578X231194808 for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo by Dimeng Song, Xiaoxue Fang, Zhiyuan Sun, Zhidong Qiu, Da Liu, Haoming Luo, Ye Jin and Donglu Wu in Natural Product Communications

Footnotes

Acknowledgments

We would like to thank Cambridge Proofreading () for English language editing and Huada Gene Company for transcriptome sequencing.

Author Contributions

The conception and design of the study: Donglu Wu, Ye Jin, and Haoming Luo. Data acquisition: Dimeng Song and Zhiyuan Sun. Data analysis and interpretation: Dimeng Song, Xiaoxue Fang, Zhidong Qiu, and Da Liu. Drafting the article or revising it critically for important intellectual content: Dimeng Song, Zhiyuan Sun, and Xiaoxue Fang. Final approval of the version to be submitted: Dimeng Song and Xiaoxue Fang. All authors have read and agreed to the published version of the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Jilin University (SY20191210; approved on 10 December 2019).

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by the National Natural Science Foundation of China (Grant No. 81903876), Jilin Provincial Department of Education (JJKH20210982KJ), and Jilin Province Youth Science and Technology Talent Support Project (2020025).

ORCID iDs

Dimeng Song

Xiaoxue Fang

Da Liu

Donglu Wu

Statement of Human Rights

No human participants were included in this study, and informed consent was not required.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not required.

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Cryptotanshinone,a Potential SIRT3 Inhibitor,Suppresses Colorectal Cancer Proliferation in vitro and in vivo

Abstract

Keywords

Introduction

Results

Transcriptome Sequencing

CPT Inhibits the Proliferation of SW480 and SW620 in vitro and in vivo

CPT Induces CRC Cell Apoptosis and Triggers Alteration of Mitochondrial Membrane Potential

SIRT3 Overexpression is Correlated With CRC Patient Survival

CPT Targets SIRT3 and Downregulates SIRT3 Protein Expression

Discussion

Conclusions

Materials and Methods

Materials and Reagents

Animal Experiment

Cell Culture

Transcriptome Sequencing

RNA Preparation

mRNA Library Preparation

RNAdenovo Analysis Method

Functional Enrichment

CCK-8

Colony Formation

Cell Cycle

Apoptosis

Western Blot

Immunofluorescence

Molecular Docking

Statistical Analysis

Supplemental Material

sj-jpg-1-npx-10.1177_1934578X231194808 - Supplemental material for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo

Supplemental Material

sj-docx-2-npx-10.1177_1934578X231194808 - Supplemental material for Cryptotanshinone, a Potential SIRT3 Inhibitor, Suppresses Colorectal Cancer Proliferation in vitro and in vivo

Footnotes

Acknowledgments

Author Contributions

Declaration of Conflicting Interests

Ethical Approval

Funding

ORCID iDs

Statement of Human Rights

Statement of Informed Consent

Supplemental Material

References

Supplementary Material