Identification of novel caspase/autophagy‐related gene switch to cell fate decisions in breast cancers

Objectives

Caspases, a family of cysteine proteases with unique substrate specificities, contribute to apoptosis, whereas autophagy‐related genes (ATGs) regulate cytoprotective autophagy or autophagic cell death in cancer. Accumulating evidence has recently revealed underlying mechanisms of apoptosis and autophagy; however, their intricate relationships still remain to be clarified. Identification of caspase/ATG switches between apoptosis and autophagy may address this problem.

Materials and methods

Identification of caspase/ATG switches was carried out using a series of elegant systems biology & bioinformatics approaches, such as network construction, hub protein identification, microarray analyses, targeted microRNA prediction and molecular docking.

Results

We computationally constructed the global human network from several online databases and further modified it into the basic caspase/ATG network. On the basis of apoptotic or autophagic gene differential expressions, we identified three molecular switches [including androgen receptor, serine/threonine‐protein kinase PAK‐1 (PAK‐1) and mitogen‐activated protein kinase‐3 (MAPK‐3)] between certain caspases and ATGs in human breast carcinoma MCF‐7 cells. Subsequently, we identified microRNAs (miRNAs) able to target androgen receptor, PAK‐1 and MAPK‐3, respectively. Ultimately, we screened a range of small molecule compounds from DrugBank, able to target the three above‐mentioned molecular switches in breast cancer cells.

Conclusions

We have systematically identified novel caspase/ATG switches involved in miRNA regulation, and predicted targeted anti‐cancer drugs. These findings may uncover intricate relationships between apoptosis and autophagy and thus provide further new clues towards possible cancer drug discovery.

     

A novel inhibitor of ADAM17 sensitizes colorectal cancer cells to 5‐Fluorouracil by reversing Notch and epithelial‐mesenchymal transition in vitro and in vivo

Objectives

Colorectal cancer is one of the most common malignancies both in men and women. Owing to metastasis and resistance, the prognosis of colorectal cancerCRC patients remains extremely poor with chemotherapy. A disintegrin and metalloproteinase 17 (ADAM17) induces the activation of Notch pathway and contributes to the chemoresistance. This study aimed to discover a novel ADAM17 inhibitor and investigate the chemosensitization effect.

Materials and methods

Pharmacophore model, western blot and enzymatic assay were used to discover ZLDI‐8. Cell proliferation was determined by MTT and colony formation assay. Cell migratory and invasive ability were determined by wound healing scratch and transwell assay. Immunofluorescence images and western blot analysed the expression of Notch or epithelial‐mesenchymal transition (EMT) pathway markers. Xenografts were employed to evaluate the chemosensitization effect of ZLDI‐8 in vivo.

Results

We found that ZLDI‐8 cell‐specifically inhibited the proliferation of CRC, and this effect was due to abrogation of ADAM17 and Notch pathway. Meanwhile, we reported for the first time that ZLDI‐8 synergistically improved the anti‐tumour and anti‐metastasis activity of 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways. Interestingly, in vivo studies further demonstrated that ZLDI‐8 promoted the anti‐tumour effect of 5‐fluorouracil through Notch and EMT reversal.

Conclusions

A novel ADAM17 inhibitor ZLDI‐8 may be a potential chemosensitizer which sensitized CRC cells to 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways.

     

Curcumin enhances the anti‐cancer effects of Paris Saponin II in lung cancer cells

Objectives

To investigate the synergistic mechanisms of Paris Saponin II (PSII) and Curcumin (CUR) in lung cancer.

Materials and Methods

The combination changed the cellular uptake of CUR and PSII, apoptosis, cell cycle arrest and cytokine levels were analysed on different lung cancer cells.

Results

The combination displayed a synergistic anti‐cancer effect through promoting the cellular uptake of CUR on different lung cancer cells. Hoechst H33258 staining and FACS assay indicated that the combination of PSII and CUR induced cell cycle arrest and apoptosis. Western blot and cytokine antibody microarray suggested that the combination activated death receptors such as DR6, CD40/CD40L, FasL and TNF‐α to induce cancer cells apoptosis, and up‐regulated IGFBP‐1 leading to inhibition of PI3K/Akt pathway and increase of p21 and p27, which therefore induced a G2 phase arrest in NCI‐H446 cells. Meanwhile, the combination suppressed PCNA and NF‐κB pathway in 4 kinds of lung cancer cells. They activated the phosphorylation of p38 and JNK, and inhibited PI3K in NCI‐H460 and NCI‐H446 cells, enhanced the phosphorylation of JNK in NCI‐H1299 cells, and increased the phosphorylation of p38 and ERK, and suppressed PI3K in NCI‐H520 cells.

Conclusions

PSII combined with CUR had a synergistic anti‐cancer effect on lung cancer cells. These findings provided a rationale for using the combination of curcumin and PSII in the treatment of lung cancer in future.

     

Low‐intensity pulsed ultrasound induced enhanced adipogenesis of adipose‐derived stem cells

Objective

The aim of this study was to investigate effects of low‐intensity pulsed ultrasound (LIPUS) on differentiation of adipose‐derived stem cells (ASCs), in vitro.

Materials and methods

Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR‐γ1, and APN, was examined by real‐time PCR. Immunofluorescence (IF) staining was performed to test for PPAR‐γ at the protein level.

Results

Our data revealed that specific patterns of LIPUS up‐regulated levels of both PPAR‐γ1 and APN mRNA, and PPAR‐γ protein.

Conclusions

In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

     

2‐(2‐nitrobenzylidene) indolin‐3‐one compound inhibits transmembrane prostate androgen‐induced protein (TMEPAI) expression and cancer cell proliferation

Objectives

The transmembrane prostate androgen‐induced protein (TMEPAI) is aberrantly expressed in many cancer and plays a crucial role in tumourigenesis, which makes it a potential cancer therapeutic target for drug discovery.

Materials and methods

Here, we employed a firefly luciferase reporter driven by the TMEPAI gene promoter to screen for compound capable of inhibiting the expression of TMEPAI, and the effects of TMEPAI inhibitor on cancer cell proliferation were evaluated using the colony formation assay, cell cycle analysis, Ki‐67 immunofluorescence assay and EdU incorporation assay.

Results

2‐(2‐nitrobenzylidene) indolin‐3‐one (JHY‐A007‐50) was identified and shown to effectively inhibit the TMEPAI promoter activity. Further studies revealed that JHY‐A007‐50 specifically inhibited the expression of TMEPAI at both the mRNA and protein levels. Moreover, we found that JHY‐A007‐50 could inhibit cell proliferation and induce cell cycle arrest at the G1 phase. Our results showed that overexpression of TMEPAI decreased the inhibitory effects of JHY‐A007‐50 on cancer cell proliferation, and JHY‐A007‐50 did not affect the cell viability of HeLa cells knocked down of TMEPAI.

Conclusions

Taken together, these results suggest that compound JHY‐A007‐50 mediates the downregulation of TMEPAI expression and inhibits cell proliferation in cancer cells.

     

Fluoxetine induces autophagic cell death via eEF2K‐AMPK‐mTOR‐ULK complex axis in triple negative breast cancer

Objectives

Triple negative breast cancer (TNBC) is a complex and intrinsically aggressive tumour with poor prognosis, and the discovery of targeted small‐molecule drugs for TNBC treatment still remains in its infancy. In this study, we aimed to discover a small‐molecule agent for TNBC treatment and illuminate its potential mechanisms.

Materials and methods

Cell viability was detected by using methylthiazoltetrazolium (MTT) assay. Electron microscopy, GFP‐LC3 transfection, monodansylcadaverine staining and apoptosis assay were performed to determine Fluoxetine‐induced autophagy and apoptosis. Western blotting and siRNA transfection were carried out to investigate the mechanisms of Fluoxetine‐induced autophagy. iTRAQ‐based proteomics analysis was used to explore the underlying mechanisms.

Results

We have demonstrated that Fluoxetine had remarkable anti‐proliferative activities and induced autophagic cell death in MDA‐MB‐231 and MDA‐MB‐436 cells. The mechanism for Fluoxetine‐induced autophagic cell death was associated with inhibition of eEF2K and activation of AMPK‐mTOR‐ULK complex axis. Further iTRAQ‐based proteomics and network analyses revealed that Fluoxetine‐induced mechanism was involved in BIRC6, BNIP1, SNAP29 and Bif‐1.

Conclusions

These results demonstrate that Fluoxetine induces apoptosis and autophagic cell death in TNBC, which will hold a promise for the future TNBC therapy.

     

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Objectives

Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti‐cancer agent. However, the molecular mechanisms involved remain poorly understood.

Materials and methods

Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA‐β‐gal assay, western blotting and immunofluorescence were performed to determine CGN‐induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN‐induced senescence and autophagy. The anti‐tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models.

Results

We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell‐cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN‐mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti‐tumour effects in vivo.

Conclusions

Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN‐mediated anti‐cancer therapy.

     

LincRNA‐p21 suppresses development of human prostate cancer through inhibition of PKM2

Objectives

Previously, we found that long intergenic non‐coding RNA‐p21 (lincRNA‐p21) inhibited the development of human prostate cancer. However, the underlying molecular mechanisms are poorly understood. Here, we attempted to investigate the downstream targets of lincRNA‐p21 in prostate cancer.

Materials and methods

Expression of lincRNA‐p21 and PKM2 was determined by qRT‐PCR and Western blot. Lentivirus expressing shPKM2 or shCtrl was used to explore the role of PKM2 on the enhanced cell proliferation and glycolysis of lincRNA‐p21‐silenced prostate cancer cells. A xenograft mouse model was performed to investigate the effect of PKM2 suppression, glycolytic or mammalian target of rapamycin (mTOR) inhibitor on the tumorigenic capacity of lincRNA‐p21‐silenced prostate cancer cells.

Results

We revealed that lincRNA‐p21 silencing in DU145 and LNCaP cells induced up‐regulation of PKM2 and activation of glycolysis, which could be reversed by PKM2 knockdown or rapamycin treatment. We also found that the proliferation and tumorigenesis of lincRNA‐p21‐silenced prostate cancer cells were significantly inhibited after knocking down PKM2. 3‐bromopyruvate (3‐Brpa) or rapamycin treatment largely decreased the tumour burden. Importantly, PKM2 expression was inversely correlated with the lincRNA‐p21 level and the survival of prostate cancer patients.

Conclusions

We demonstrated that lincRNA‐p21 blunted the prostate cancer cell proliferation and tumorigenic capacity through down‐regulation of PKM2. Therefore, targeting PKM2 or glycolysis might be a therapeutic strategy in prostate cancer patients with lowly expressed lincRNA‐p21.

     

Mesenchymal stem cell transfusion for desensitization of positive lymphocyte cross‐match before kidney transplantation: outcome of 3 cases

Objectives

Donor specific antibodies (DSA) and a positive cross‐match are contraindications for kidney transplantation. Trials of allograft transplantation across the HLA barrier have employed desensitization strategies, including the use of plasmapheresis, intravenous immunoglobulins, anti‐B‐cell monoclonal antibodies and splenectomy, associated with high‐intensity immunosuppressive regimens. Our case 1 report suffered from repeatedly positive lymphocyte cross match after 1st renal transplantation. Graft nephrectomy could not correct the state of sensitization. Splenectomy was done in a trial to get rid of the antibody producing clone. Furthermore plasmapheresis with low dose IVIG could not as well revert the state of sensitization for the patient.

Material and methods

About 50 millions donor specific MSCs were injected to the patient.

Results

MSCs transfusion proved to be the only procedure which could achieve successful desensitization before performing the second transplantation owing to their immunosuppressive properties.

Conclusion

This case indicates that DS‐MSCs is a potential option for anti‐HLA desensitization. In cases 2 and 3 IV DS‐MSCs transfusion was selected from the start as a successful line of treatment for pre renal transplantation desensitization to save other unnecessary lines of treatment that were tried in case 1.

     

Comprehensive molecular profiling of the B7 family in gastrointestinal cancer

Objectives

B7 family has been identified as co‐stimulatory or co‐inhibitory molecules on T‐cell response and plays an important role in tumour mortality and malignancy. In this study, the expression pattern of B7 family in gastrointestinal (GI) cancer was examined. Its upstream regulating mechanism, downstream targets and association with clinical parameters were also studied.

Materials and methods

The expression level of B7 members was analysed by FIREHOUSE. The gene mutation, DNA methylation, association with clinical parameters and downstream network of B7 members were analysed in cBioportal. The mutation frequency was analysed by Catalogue of Somatic Mutations in Cancer (COSMIC) analysis. The phylogenetic tree was constructed in MEGA7. The interaction protein domain analysis was performed by Pfam 31.0.

Results

Differential expression of B7 family molecules was detected in different kinds of GI cancer. High‐frequency gene alteration was found in tumour samples. There was negative correlation of promoter methylation and mRNA expression of B7 family members in tumour samples, suggesting the epigenetic basis of B7 family gene deregulation in GI cancer. The overexpression of B7‐H1 in pancreatic cancer, B7‐H5 in oesophageal cancer and B7‐H6 in liver cancer were significantly associated with worse overall survival. Finally, by network analysis, we identified some potential interacting proteins for B7‐1/2 and B7‐H1/DC.

Conclusions

Overall, our study suggested that B7 member deregulation was strongly involved in GI cancer tumorigenesis.

     

Effects of simvastatin and rosuvastatin on RAS protein,matrix metalloproteinases and NF‐κB in lung cancer and in normal pulmonary tissues

Objectives

In this study, we have evaluated effects of 24‐hour treatments with simvastatin or rosuvastatin on RAS protein, NF‐κB and MMP expression in LC tissues obtained from 12 patients undergoing thoracic surgery.

Materials and methods

Normal and lung tumour tissues obtained from each sample were exposed to simvastatin (2.5–30 μm ) or rosuvastatin (1.25–30 μm ) and western blot analysis was then performed.

Results

We documented increased expression of proteins, MMP‐2, MMP‐9 and NF‐κB‐p65 in LC tissues, with respect to normal tissues (P < 0.01). In the malignant tissues, simvastatin and rosuvastatin significantly (P < 0.01) and dose‐dependently reduced RAS protein, MMP‐2/9 and NF‐κB‐p65 expression.

Conclusions

In conclusion, our results suggest that simvastatin and rosuvastatin could play a role in LC treatment by modulation of RAS protein, MMP‐2/9 and NF‐κB‐p65.

     

Berberine inhibits the chemotherapy‐induced repopulation by suppressing the arachidonic acid metabolic pathway and phosphorylation of FAK in ovarian cancer

Objectives

Cytotoxic chemotherapy is an effective and traditional treatment of ovarian cancer. However, chemotherapy‐induced apoptosis may also trigger and ultimately accelerate the repopulation of the small number of adjacent surviving cells. This study mainly focused on the tumour cell repopulation caused by chemotherapy in ovarian cancer and the adjunctive/synergistic effect of Berberine on the prevention of tumour repopulation.

Materials and methods

The transwell system was used to mimic the co‐culture of surviving ovarian cancer cells in the microenvironment of cytotoxic chemotherapy‐treated dying cells. Tumour cell proliferation was observed by crystal violet staining. AA and PGE₂ levels were measured by ELISA, and changes of protein expression were analysed by Western blot.

Results

Chemotherapy drug VP16 treatment triggered AA pathway, leading to the elevated PGE₂ level, and ultimately enhanced the repopulation of ovarian cancer cells. Berberine can block the caspase 3‐iPLA₂‐AA‐COX‐2‐PGE₂ pathway by inhibiting the expression of iPLA₂ and COX‐2. Berberine can also reverse the increased phosphorylation of FAK caused by abnormal PGE₂ level and thus reverse the repopulation of ovarian cancer cells after VP16 treatment.

Conclusions

Our observation suggested that Berberine could inhibit the chemotherapy‐induced repopulation of ovarian cancer cells by suppressing the AA pathway and phosphorylation of FAK. And these findings implicated a novel combined use of Berberine and chemotherapeutics, which might prevent ovarian cancer recurrence by abrogating early tumour repopulation.

     

Critical role of inflammatory mast cell in fibrosis: Potential therapeutic effect of IL‐37

Background

Fibrosis involves the activation of inflammatory cells, leading to a decrease in physiological function of the affected organ or tissue.

Aims

To update and synthesize relevant information concerning fibrosis into a new hypothesis to explain the pathogenesis of fibrosis and propose potential novel therapeutic approaches.

Materials and Methods

Literature was reviewed and relevant information is discussed in the context of the pathogenesis of fibrosis.

Results

A number of cytokines and their mRNA are involved in the circulatory system and in organs of patients with fibrotic tissues. The profibrotic cytokines are generated by several activated immune cells, including fibroblasts and mast cells (MCs), which are important for tissue inflammatory responses to different types of injury. MC‐derived TNF, IL‐1, and IL‐33 contribute crucially to the initiation of a cascade of the host defence mechanism(s), leading to the fibrosis process. Inhibition of TNF and inflammatory cytokines may slow the progression of fibrosis and improve the pathological status of the affected subject. IL‐37 is generated by various types of immune cells and is an IL‐1 family member protein. IL‐37 is not a receptor antagonist; it binds IL‐18 receptor alpha (IL‐18Rα) and delivers the inhibitory signal by using TIR8. It has been shown that IL‐37 can be protective in inflammation and injury, and inhibits both innate and adaptive immunity.

Discussion

IL‐37 may be useful for suppression of inflammatory diseases induced by inhibiting MyD88‐dependent TLR signalling. In addition, IL‐37 downregulates NF‐κB induced by TLR2 or TLR4 through a mechanism dependent on IL‐18Rα.

Conclusion

This review summarizes current knowledge on the role of MC in inflammation and tissue/organ fibrosis, with a focus on the therapeutic potential of IL‐37‐targeting cytokines.

     

Low level laser therapy induces increased viability and proliferation in isolated cancer cells

Objectives

Low level laser therapy (LLLT), which stimulates natural biological processes in the application region, is frequently used in dental treatments. The aim of our study was to evaluate the effects of LLLT which could activate precancerous cells or increase existing cancerous tissue in case of clinically undetectable situations.

Materials and methods

Saos‐2 osteoblast‐like osteosarcoma cells and A549 human lung carcinoma cells were used. Twenty‐four hours after preparation of cell culture plates, laser irradiation was performed 1, 2 and 3 times according to the test groups using Nd:YAG laser with the power output 0.5, 1, 2 and 3 W. Cell proliferation analysis was performed by MTT assay at the 24th hour following the last laser applications.

Results

Generally, it was observed that the proliferation rates increased as the number of applications increased, when compared to the controls, especially in those cases in which the irradiation was performed 2 or 3 times more.

Conclusion

The findings of this study have led to the conclusion that LLLT increases cancer cell proliferation, depending on the power output level of the laser and the number of applications. In addition to the proliferation and mitotic activity of the cancer tissue cells, we concluded that LLLT, which is frequently used in dental practice, could activate precancerous cells or increase existing cancerous tissue.

     

Roflumilast enhances cisplatin‐sensitivity and reverses cisplatin‐resistance of ovarian cancer cells via cAMP/PKA/CREB‐FtMt signalling axis

Objective

We previously demonstrated the roflumilast inhibited cell proliferation and increased cell apoptosis in ovarian cancer. In this study, we aimed to investigate the roles of roflumilast in development of cisplatin (DDP)‐sensitive and ‐resistant ovarian cancer.

Methods

OVCAR3 and SKOV3 were selected and the corresponding DDP‐resistant cells were constructed. Cell viability, proliferation, apoptosis, cycle were performed. Expression cAMP, PKA, CREB, phosphorylation of CREB and FtMt were detected. The roles of roflumilast in development of DDP‐sensitive and ‐resistant ovarian cancer were confirmed by xenograft model.

Results

Roflumilast + DDP inhibited cell proliferation, and induced cell apoptosis and G0/G1 arrest in OVCAR3 and SKOV3 cells, roflumilast induced expression of FtMt, the activity of cAMP and PKA and phosphorylation of CREB in ovarian cancer cells and the above‐effect were inhibited by H89. Downregulation of CREB inhibited the roflumilast‐increased DDP sensitivity of ovarian cancer cells, and the roflumilast‐induced FtMt expression and phosphorylation of CREB. Also, roflumilast reversed cisplatin‐resistance, and induced expression of FtMt and activation of cAMP/PKA/CREB in DDP‐resistant ovarian cancer cells. Similarly, treated with H89 or downregulation of CREB inhibited the changes induced by roflumilast. In vivo, roflumilast inhibited the development of SKOV3 or SKOV3‐DDP‐R xenograft models.

Conclusions

Roflumilast enhanced DDP sensitivity and reversed the DDP resistance of ovarian cancer cells via activation of cAMP/PKA/CREB pathway and upregulation of the downstream FtMt expression, which has great promise in clinical treatment.

     

The role of Sprouty1 in the proliferation,differentiation and apoptosis of epidermal keratinocytes

Objectives

Sprouty (SPRY) 1 is one of the SPRY proteins that inhibits signalling from various growth factors pathways and has also been known as a tumour suppressor in various malignancies. However, no study elucidates the role of SPRY1 in the skin. Our study was conducted to determine the function of SPRY1 in human keratinocytes and the epidermis.

Materials and methods

In vitro primary cultured epidermal keratinocytes were used to investigate the proliferation, differentiation and apoptosis of these cells. We also established overexpression of SPRY1 in vitro and K14‐SPRY1 transgenic mice.

Results

SPRY1 was mainly located in the cytoplasm of the epidermal keratinocytes from the granular epidermal layer of the skin and cultured cells. Overexpressed SPRY1 in keratinocytes resulted in up‐regulation of P21, P27 and down‐regulation of cyclin B1; decrease in MMP3 and integrin α6. SPRY1‐overexpressed primary keratinocytes exhibited a lower proliferation and migration capability and higher rates of apoptosis. Epidermis of SPRY1‐TG mice represented delayed wound healing. Proteomics analysis and GO enrichment showed DEPs of SPRY1 TG mice epidermis is significantly enriched in immune‐ and inflammatory‐associated biological process.

Conclusions

In summary, SPRY1 expression was inversely correlated with cell proliferation, migration and promote cell apoptosis of keratinocytes. SPRY1 maybe a negative feedback regulator in normal human epidermal keratinocytes and cutaneous inflammatory responses. Our study raised the possibility that enhancing expression of SPRY1 may have the potential to promote anti‐inflammatory effects.

     

Effect of substrate stiffness on proliferation and differentiation of periodontal ligament stem cells

Objectives

The aim of this study was to understand the effect of substrate stiffness (a mechanical factor of the extracellular matrix) on periodontal ligament stem cells (PDLSCs) and its underlying mechanism.

Materials and methods

Elastic substrates were fabricated by mixing 2 components, a base and curing agent in proportions of 10:1, 20:1, 30:1 or 40:1. PDLSC morphology was observed using scanning electron microscopy (SEM). Cell proliferation and differentiation were assessed after PDLSCs was cultured on various elastic substrates. Data were analysed using one‐way ANOVA.

Results

SEM revealed variations in the morphology of PDLSCs cultured on elastic substrates. PDLSC proliferation increased with substrate stiffness (P < .05). Osteogenic differentiation of PDLSCs was higher on stiff substrates. Notch pathway markers were up‐regulated in PDLSCs cultured on stiff substrates.

Conclusions

Results suggested that the osteogenic differentiation of PDLSCs might be promoted by culturing them in a stiffness‐dependent manner, which regulates the Notch pathway. This might provide a new method of enhancing osteogenesis in PDLSCs.