Long non-coding RNAs in cancer: multifaceted roles and potential targets for immunotherapy.

Cancer remains a major global health concern with high mortality rates mainly due to late diagnosis and poor prognosis. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression in human cancer, functioning through various mechanisms including as competing endogenous RNAs (ceRNAs) and indirectly regulating miRNA expression. LncRNAs have been found to have both oncogenic and tumor-suppressive roles in cancer, with the former promoting cancer cell proliferation, migration, invasion, and poor prognosis. Recent research has shown that lncRNAs are expressed in various immune cells and are involved in cancer cell immune escape and the modulation of the tumor microenvironment, thus highlighting their potential as targets for cancer immunotherapy. Targeting lncRNAs in cancer or immune cells could enhance the anti-tumor immune response and improve cancer immunotherapy outcomes. However, further research is required to fully understand the functional roles of lncRNAs in cancer and the immune system and their potential as targets for cancer immunotherapy. This review offers a comprehensive examination of the multifaceted roles of lncRNAs in human cancers, with a focus on their potential as targets for cancer immunotherapy. By exploring the intricate mechanisms underlying lncRNA-mediated regulation of cancer cell proliferation, invasion, and immune evasion, we provide insights into the diverse therapeutic applications of these molecules.

7-amino carboxycoumarin 2 inhibits lactate induced epithelial-to-mesenchymal transition via MPC1 in oral and breast cancer cells.

Lactate is an oncometabolite that play important role in tumor aggressiveness. Lactate from the tumor microenvironment (TME) is taken up by cancer cells as an energy resource via mitochondrial oxidative phosphorylation (or OXPHOS). In the present study, by using an online meta-analysis tool we demonstrated that in oral squamous cancer cells (OSCCs) glycolytic and OXPHOS governing genes are overexpressed, like in breast cancer. For experimental demonstration, we treated the OSCC cell line (SCC4) and breast cancer cells (MDA-MB-231) with sodium L-lactate and analyzed its effects on changes in EMT and migration. For the therapeutic intervention of lactate metabolism, we used AZD3965 (an MCT1 inhibitor), and 7ACC2 (an MPC inhibitor). Like breast cancer, oral cancer tissues showed increased transcripts of 12 genes that were previously shown to be associated with glycolysis and OXPHOS. We experimentally demonstrated that L-lactate treatment induced mesenchymal markers and migration of cancer cells, which was significantly neutralized by MPC inhibitor that is, 7ACC2. Such an effect on EMT status was not observed with AZD3965. Furthermore, we showed that lactate treatment increases the MPC1 expression in both cancer cells, and this might be the reason why cancer cells in the high lactate environment are more sensitive to 7ACC2. Overall, our present findings demonstrate that extracellular lactate positively regulates the MPC1 protein expression in cancer cells, thereby putting forward the notion of using 7ACC2 as a potential therapeutic alternative to inhibit malignant oxidative cancers. Future preclinical studies are warranted to validate the present findings.

Clinical significance of Notch pathway-associated microRNA-107 in pancreatic ductal adenocarcinoma.

Background & aim: MicroRNAs associated with the Notch pathway play a critical role in the progression of pancreatic carcinoma. Our aim was to study the clinical significance of miR-107 and NOTCH2 in pancreatic ductal adenocarcinoma (PDAC). Methods: The circulating miR-107 levels in PDAC and controls were determined by qPCR. NOTCH2 protein (target) expression in tissue of PDAC, periampullary carcinoma, chronic pancreatitis and normal pancreatic tissue was assessed by immunohistochemistry. Results: The circulating miR-107 levels were found to be significantly reduced in PDAC as compared with controls. Additionally, NOTCH2 protein expression was higher in PDAC tissue as compared with controls and was clinically associated with metastasis. Conclusion: Our findings demonstrate the utility of circulating miR-107 as a potential differentiating marker in PDAC.

The Expression of the RUVBL1 Component of the R2TP Complex Correlates with Poor Prognosis in DLBCL.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin's lymphoma (NHL) accounting for 30% of adult NHL worldwide and 50% in developing countries like India. DNA damage and Myc-induced transformation are well-known contributing factors towards development of DLBCL. A recently identified HSP90 co-chaperone complex R2TP has been shown to contribute towards DNA damage and Myc-induced transformation. This study aimed to analyse the immunohistochemical (IHC) expression of R2TP complex components RUVBL1, PIH1D1, and RPAP3 in DLBCL patients and correlate with prognosis. METHODS: DLBCL (n = 54) histological slides were retrieved from archives, and detailed histomorphological and clinical features were noted. IHC staining of R2TP complex components RUVBL1, PIH1D1, and RPAP3 was performed on 54 cases (FFPE) of DLBCL. Expression data were correlated with survival and clinical features. RESULTS: Out of the 54 DLBCL cases, 59.26% (n = 32) stained positive for RUVBL1. The RUVBL1 expression was associated with poor prognosis in both progression-free survival (PFS) (p = 0.0146) and overall survival (OS) (p = 0.0328). The expression was positively correlated with bone marrow involvement (p = 0.0525). The expression of PIH1D1 was observed in 68.51% (n = 32) of DLBCL cases, and positive correlation was observed with international prognostic index score (p = 0.0246); however, no correlation was observed with PFS or OS. Finally, RPAP3 was found immunopositive in only 1 case of DLBCL. CONCLUSIONS: Immunopositivity for RUVBL1 is associated with poor prognosis along with a higher relapse rate amongst the DLBCL patients. PIH1D1 immunopositivity correlated with a higher IPI score.

Expression pattern, regulation, and clinical significance of TOX in breast cancer.

Thymocyte selection-associated high mobility group box protein (TOX) is a transcription factor implicated in the regulation of T cell exhaustion during chronic infection and cancer. While TOX is being targeted for cancer immunotherapy, limited information is available about its significance in breast cancer and other solid tumors. We performed a comprehensive analysis of TOX gene expression, its epigenetic regulation, protein localization, relation to tumor infiltrating immune cell composition, and prognostic significance in breast cancer using publicly available datasets. Our results suggest an inverse correlation between TOX expression and DNA methylation in tumor cells. However, its expression is elevated in tumor infiltrating immune cells (TIICs), which may compensates for the total TOX levels in the tumor as a whole. Furthermore, higher TOX levels in tumors are associated with T cell exhaustion signatures along with presence of active inflammatory response, including elevated levels of T cell effector cytokines. Survival analysis also confirmed that higher expression of TOX is associated with better prognosis in breast cancer. Therefore, expression of TOX may serve as a novel prognostic marker for this malignancy.

A glycoprotein from mammary gland secreted during involution promotes apoptosis: Structural and biological studies.

Secretory signalling glycoprotein (SPX-40) from mammary gland is highly expressed during involution. This protein is involved in a programmed cell death during tissue remodelling which occurs at the end of lactation. SPX-40 was isolated and purified from buffalo (SPB-40) from the samples obtained during involution. One solution of SPB-40 was made by dissolving it in buffer containing 25 mM Tris-HCl and 50 mM NaCl at pH 8.0. Another solution was made by adding 25% ethanol to the above solution. The biological effects of SPB-40 dissolved in above two solutions were evaluated on MCF-7 breast cancer cell lines. Free SPB-40 indicated significant pro-apoptotic effects while ethanol exposed SPB-40 showed considerably reduced effects on the apoptosis. SPB-40 was crystallized in the native state. The crystals of SPB-40 were soaked in four separate solutions containing 25% acetone, 25% ethanol, 25% butanol and 25% MPD. Four separate data sets were collected and their structures were determined at high resolutions. In all the four structures, the molecules of acetone, ethanol, butanol and MPD respectively were observed in the hydrophobic binding pocket of SPB-40. As a result of which, the conformation of Trp78 was altered thus blocking the binding site in SPB-40 leading to the loss of activity.

Clinical significance of cathepsin L and cathepsin B in dilated cardiomyopathy.

Dysregulated expression of lysosomal cysteine cathepsins is associated with adverse cardiac remodeling, a characteristic of several cardiovascular diseases. However, the information regarding the role of cysteine cathepsin L (CTSL) and cathepsin B (CTSB) in dilated cardiomyopathy (DCM) is limited. The present study was aimed to investigate the expression of CTSL and CTSB in animal model of doxorubicin (doxo)-induced cardiomyopathy as well as in peripheral blood samples of DCM patients. Cardiac tissue sections from doxo-treated and control rats were used to study the expression of CTSL and CTSB by enzyme assay and immunohistochemistry (IHC). Peripheral blood mononuclear cells (PBMCs) isolated from DCM patients (n = 29) along with age-matched healthy controls (n = 28) were used to assay enzymatic activity of these cathepsins. Activities of these proteases were further correlated with echocardiographic parameters of DCM patients. A significant increase in CTSL activity and protein expression was observed with no changes in CTSB levels in doxo-treated rats as compared to controls. We also observed a drastic increase in the functional activity of cathepsin L+cathepsin B (CTSL+B), CTSL, and CTSB in DCM patients compared to controls (p ≤ 0.001). Increased levels of these proteases exhibited a statistically significant correlation with reduced left ventricular ejection fraction (LVEF) in DCM patients (ρ = -0.58, p = 0.01). For the first time, this study demonstrates a correlation between increased expression of CTSL and CTSB in PBMCs with severity of left ventricular dysfunction in DCM patients. Thus, these proteases may serve as blood-based biomarker of DCM and prove useful in its management.

Prognostic significance of plasma matrix metalloprotease-2 in pancreatic cancer patients.

BACKGROUND & OBJECTIVES: Pancreatic cancer has a propensity for wide stromal invasion. Matrix metalloprotease-2 (MMP-2) is a protease that degrades the peri-tumoural tissue and helps in tumour dissemination. Thus, this study was aimed to assess any association of plasma MMP-2 levels with clinicopathological parameters and survival of patients with pancreatic cancer. METHODS: Plasma samples from 127 pancreatic cancer patients were analyzed for MMP-2 levels by ELISA. Survival and other clinicopathological parameters of patients were analyzed for any correlation with plasma MMP-2 levels. RESULTS: The mean MMP-2 levels in pancreatic cancer patients were 560.3±222.0 ng/ml which were significantly elevated compared to chronic pancreatitis patients (P<0.001) and healthy individuals (P<0.05). The plasma levels of MMP-2 significantly correlated with tissue expression of this protease (P=0.004). However, MMP-2 levels did not exhibit any association either with clinicopathological parameters or with survival. INTERPRETATION & CONCLUSIONS: Elevated MMP-2 levels were observed in blood of pancreatic cancer patients which correlated with its tissue expression. However, these levels did not associate with survival or any clinicopathological parameters of patients. Further studies need to be done to confirm the prognostic/ clinical significance of MMP-2 in cancer patients before and after surgery.

Human dipeptidyl peptidase III regulates G-protein coupled receptor-dependent Ca2+ concentration in human embryonic kidney 293T cells.

The precise biological function of human dipeptidyl peptidase III (hDPP III) is poorly understood. Using luciferase reporter constructs responsive to change in Ca2+ and/or cAMP and Fura 2-AM fluorometric assay, we show a significant decrease in intracellular Ca2+ following hDPP III overexpression and angiotensin II stimulation in angiotensin II type 1 receptor (G-protein coupled receptor, GPCR) expressing HEK293T cells. Silencing the expression of hDPP III by siRNA reversed the effect of hDPP III overexpression with a concomitant increase in Ca2+. These results, for the first time, show involvement of hDPP III in GPCR dependent Ca2+ regulation in HEK293T cells.

Human dipeptidyl peptidase III mRNA variant I and II are expressed concurrently in multiple tumor derived cell lines and translated at comparable efficiency in vitro.

Dipeptidyl peptidase III (DPP III) is an emerging biomarker of human cancers. Expression, specificity, and function of human DPP III (hDPP III) mRNA variant I (V-I), II (V-II), and III (V-III) are poorly understood. Here, we investigated expression of these variants in multiple human tumor derived cell lines. DNA sequencing revealed concurrent expression of hDPP III V-I and V-II in U87MG (glioblastoma), SCC4 (squamous cell carcinoma), SiHa (carcinoma of uterus) cells. In SKOV1 cells, a cell line derived from ovarian carcinoma where a positive correlation between histological aggressiveness of the malignancy and hDPP III expression has previously been established, only V-II could be detected. Human DPP III V-III, which lacks an in-frame coding sequence, could not be detected in any of these cell lines. 5' untranslated region (UTR) of hDPP III V-II contains nucleotides GCA (-12 to -10 bp) upstream to the translation initiator codon (AUG). These nucleotides are absent from V-I and V-III, however, both V-I and V-II encode for the same hDPP III protein isoform-I. In vitro transcription coupled translation assay using hDPP III V-I and V-II expression vectors which contained full length V-I and V-II cDNA including the variable 5' UTR cloned under T7 promoter, respectively revealed a comparable translational efficiency for both the variants, abrogating involvement of nucleotides GCA (-12 to -10 bp) in translation of the variants. Our results, for the first time, demonstrate concurrent expression in multiple tumor derived cell lines and a comparable in vitro translational efficiency for hDPP III V-I and II.

High levels of cell-free circulating nucleic acids in pancreatic cancer are associated with vascular encasement, metastasis and poor survival.

Pancreatic cancer is a highly aggressive disease with rapid invasion and early encasement of blood vessels. Hence, levels of circulating nucleic acids and tumor-associated mutations in them may have clinical importance. We analyzed the levels of circulating tumor DNA and oncogenic k-ras mutation in plasma of patients with pancreatic cancer and correlated their levels with survival and clinicopathological parameters. Higher levels of plasma DNA (>62 ng/mL) was found to associate significantly with lower overall survival time (p=.002), presence of vascular encasement (p=.030) and metastasis (p=.001). However, k-ras mutation status did not correlate with any of the clinicopathological parameters or survival. We conclude that circulating DNA in plasma can be an important predictor of prognosis in pancreatic cancer.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis.

BACKGROUND: Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. METHODS: Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. RESULTS: MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. CONCLUSIONS: Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC.

Clinical Significance of MUC4 and Associated Proteins in Pancreatic and Periampullary Cancers.

OBJECTIVE: This study primarily aimed to assess the expression of MUC4 in patients with pancreatic ductal adenocarcinoma (PDAC) as compared with controls and assess its clinical relevance. MATERIALS AND METHODS: Serum MUC4 levels and MUC4 gene expression in snap-frozen tissue were analyzed through surface plasmon resonance and quantitative polymerase chain reaction, respectively. Tumor tissues and control tissues were analyzed for MUC4 and other mucins through immunohistochemistry. RESULT: MUC4 expression in tumor tissue was found to be significantly elevated in PDAC patients as compared with chronic pancreatitis tissues and normal pancreatic tissues. Periampullary carcinoma and cholangiocarcinoma tissue also showed increased expression of MUC4 and other mucins. CONCLUSIONS: Differential expression of MUC4 in pancreatic tumor tissues can help to differentiate PDAC from benign conditions.

MicroRNA Signatures for Pancreatic Cancer and Chronic Pancreatitis: Expression Profiling by NGS.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease due to the lack of early detection. Because chronic pancreatitis (CP) patients are a high-risk group for pancreatic cancer, this study aimed to assess the differential miRNA profile in pancreatic tissue of patients with CP and pancreatic cancer. METHODS: MiRNAs were isolated from formalin-fixed paraffin-embedded pancreatic tissue of 22 PDAC patients, 18 CP patients, and 10 normal pancreatic tissues from autopsy (C) cases and processed for next-generation sequencing. Known and novel miRNAs were identified and analyzed for differential miRNA expression, target prediction, and pathway enrichment between groups. RESULTS: Among the miRNAs identified, 166 known and 17 novel miRNAs were found exclusively in PDAC tissues, while 106 known and 10 novel miRNAs were found specifically in CP tissues. The pathways targeted by PDAC-specific miRNAs and differentially expressed miRNAs between PDAC versus CP tissues and PDAC versus control tissues were the proteoglycans pathway, Hippo signaling pathway, adherens junction, and transforming growth factor-ß signaling pathway. CONCLUSIONS: This study resulted in a set of exclusive and differentially expressed miRNAs in PDAC and CP can be assessed for their diagnostic value. In addition, studying the role of miRNA-target gene interactions in carcinogenesis may open new therapeutic avenues.

Prognostic significance of extracellular matrix degrading enzymes-cathepsin L and matrix metalloproteases-2 [MMP-2] in human pancreatic cancer.

In the present study, we assessed the expression of extracellular matrix (ECM) degrading proteases-cathepsin L and matrix metalloprotease-2 (MMP-2) in pancreatic cancer tissue and correlated their levels with clinicopathological parameters and survival. Both the proteases were expressed in the majority of the tumor tissues examined. Staining intensity of cathepsin L was significantly higher in the tumor stroma compared to tumor epithelium while MMP-2 staining showed no such difference. Both proteases showed correlation with some of the clinicopathological parameters but only cathepsin L expression in tumor epithelium predicted a poor prognosis for the disease.

Quantitative Evaluation of Pentraxin-3 in Peri-Miniscrew Implant Crevicular Fluid in Patients Undergoing Orthodontic Treatment: A Prospective Study.

OBJECTIVE: To assess the levels of Pentraxin-3 (PTX3) in peri-miniscrew implant crevicular fluid (PMICF) before and after orthodontic force application Material and Methods: This study included 40 miniscrew implants (MSI) sites in 11 orthodontic patients with high arch discrepancy requiring first premolar extraction using maximum anchorage mechanics for the retraction of anterior teeth. After alignment, the en-masse anterior retraction was carried out using the MSI-supported direct anchorage method. PMICF was collected from the crevice of MSI using Periopaper strips 1.2µl (Oraflow Inc. USA) after one hour, 24 hours, and three weeks of MSI insertion and after one hour, 24 hours, seven days, three weeks, and six weeks of the force application. Samples were quantitatively analyzed for PTX3 levels through enzyme-linked immunosorbent assay (ELISA). RESULTS: The trend in the change of PTX3 levels was evaluated using the Wilcoxon signed-rank test. The mean concentration of PTX3 immediately after MSI insertion was 1.19 ng/ml, significantly higher than after 3 weeks after MSI insertion (0.72 ng/ml), which may correspond to the baseline. After loading, the mean PTX3 concentration increased significantly with the peak at 24 hrs (1.28 ng/ml), followed by a gradual decline till the completion of the study (0.5 ng/ml). CONCLUSION: After MSI insertion, a rise in PTX3 levels in PMICF suggests an underlying inflammatory process. The slow decline in PTX3 level and return to the baseline after loading suggests an adaptive bone response to the stimulus.

Involvement of DPP3 in modulating oncological features and oxidative stress response in esophageal squamous cell carcinoma.

Resistance to therapy in esophageal squamous cell carcinoma (ESCC) is a critical clinical problem and identification of novel therapeutic targets is highly warranted. Dipeptidyl peptidase III (DPP3) is a zinc-dependent aminopeptidase and functions in the terminal stages of the protein turnover. Several studies have reported overexpression and oncogenic functions of DPP3 in numerous malignancies. The present study aimed to determine the expression pattern and functional role of DPP3 in ESCC. DPP3 expression was assessed in normal and tumor tissues using quantitative real-time (qRT)-PCR and corroborated with ESCC gene expression datasets from Gene Expression Omnibus (GEO) and The cancer genome atlas (TCGA). DPP3 stable knockdown was performed in ESCC cells by shRNA and its effect on cell proliferation, migration, cell cycle, apoptosis, and activation of nuclear factor erythroid 2-related factor 2 (NRF2) pathway was assessed. The results suggested that DPP3 is overexpressed in ESCC and its knockdown leads to reduced proliferation, increased apoptosis, and inhibited migration of ESCC cells. Additionally, DPP3 knockdown leads to down-regulation of the NRF2 pathway proteins, such as NRF2, G6PD, and NQO1 along with increased sensitivity toward oxidative stress-induced cell death and chemotherapy. Conclusively, these results demonstrate critical role of DPP3 in ESCC and DPP3/NRF2 axis may serve as an attractive therapeutic target against chemoresistance in this malignancy.

CXCR7 mediated Giα independent activation of ERK and Akt promotes cell survival and chemotaxis in T cells.

Chemokine receptors CXCR7 and CXCR4 bind to the same ligand stromal cell derived factor-1alpha (SDF-1α/CXCL12). We assessed the downstream signaling pathways mediated by CXCL12-CXCR7 interaction in Jurkat T cells. All experiments were carried out after functionally blocking the CXCR4 receptor. CXCL12, on binding CXCR7, induced phosphorylation of extra cellular regulated protein kinases (ERK 1/2) and Akt. Selective inhibition of each signal demonstrated that phosphorylated ERK 1/2 is essential for chemotaxis and survival of T cells whereas activation of Akt promotes only cell survival. Another interesting finding of this study is that CXCL12-CXCR7 interaction under normal physiological conditions does not activate the p38 pathway. Furthermore, we observed that the CXCL12 signaling via CXCR7 is Giα independent. Our findings suggest that CXCR7 promotes cell survival and does not induce cell death in T cells. The CXCL12 signaling via CXCR7 may be crucial in determining the fate of the activated T cells.

Evaluation of Heterogeneous Nuclear Ribonucleoprotein D Expression as a Diagnostic Marker for Oral Squamous Cell Carcinoma.

The heterogeneous nuclear ribonucleoprotein D (hnRNPD) serves as a prognostic marker for oral squamous cell carcinoma (OSCC). We evaluated the diagnostic potential of hnRNPD to differentiate between OSCC and normal mucosa. Immunohistochemistry for hnRNPD and a routinely used diagnostic marker deltaNp63 (p40) was performed in 32 normal mucosae and 46 OSCC specimens. Subsequently, receiver-operating characteristic analysis was performed to evaluate the diagnostic potential of hnRNPD in comparison to that of p40. Immunostaining for p40 and hnRNPD was observed in 39 (84.78%) and 38 (82.60%) cases, respectively, in OSCC specimens. The poorly differentiated squamous cell carcinoma displayed 100% (eight cases) immunoreactivity for hnRNPD as compared to 87.5% (seven cases) for p40. Nuclear staining of p40 and hnRNPD was observed in all OSCC specimens. p40 staining was restricted to basal cells, whereas both basal and para-basal cells displayed hnRNPD staining in OSCC specimens. Areas under the curve for p40 and hnRNPD were 0.86 and 0.87, respectively. p40 and hnRNPD showed equal sensitivities (80.95%). However, hnRNPD displayed marginally higher (88.23%) specificity for tumor cells as compared to that of p40 (85.29%). Conclusion: In addition to being a well-established prognostic marker, hnRNPD can serve as a diagnostic marker for OSCC.

Effects of submucosally administered platelet-rich plasma on the rate of tooth movement.

OBJECTIVES: To evaluate the effects of submucosally administered platelet-rich plasma (PRP) on the rate of maxillary canine retraction. Levels of soluble receptor activator of nuclear factor-κb ligand (sRANKL) and osteoprotegerin (OPG) in the gingival crevicular fluid (GCF) were also measured over 2 months. MATERIALS AND METHODS: This split-mouth trial involved 20 sites in 10 subjects randomly assigned to PRP (experimental) side and control side. After alignment, the freshly prepared PRP was injected submucosally distal to the experimental side maxillary canine, and retraction was performed using NiTi closed-coil springs (150 g) on 0.019 × 0.025-inch stainless steel wire. The rate of canine movement was assessed using digital model superimposition at 0, 30, and 60 days. The OPG and sRANKL were assayed using enzyme-linked immunosorbent assay from GCF collected at 0, 1, 7, 21, 30, and 60 days. RESULTS: Twenty sites were analyzed using paired t test. The rate of tooth movement increased significantly by 35% on the PRP side compared with the control side in the first month (P = .0001) and by 14% at the end of the second month (P = .015). Using the Mann-Whitney U test, OPG levels were found to be significantly decreased on the 7th (P = .003) and 30th day on the PRP side (P = .01), while sRANKL became detectable by the third week postinjection on the PRP side (P = .069). CONCLUSIONS: Submucosal injection of platelet-rich plasma significantly increased tooth movement during the 60-day observation period. Local injection of PRP significantly altered the levels of OPG and sRANKL in GCF.