| PART ONE The correlation study of calcium metabolism and bone metastases of lung cancerBackground and purposeBone is the most common site of metastatic spread in advanced solid tumors. Almost 65%?80% patients with breast cancer, prostate cancer, and 30%?40% patients with lung cancer will develop bone metastases in the disease progresses, in particular, the incidence rate of bone metastasis could reach 60% to 80% in lung cancer patients. Patients with bone metastases present with severe bone pain, high incidence of hypercalcemia and various complications. Patients with bone metastases always have high serum calcium levels because calcium releasing caused by osteolytic metastatic carcinoma from bone matrix into the bloodstream exceed calcium excretion through by intestines and kidneys.1,25-dihydroxyvitamin D(1,25 (OH)2D3), the hormonal derivative of vitamin D, is an essential factor to balance the calcium and phosphorous in blood and participate bone metabolic regulation. Oncology patients usually have suboptimal levels of 1,25(OH)2D3 as a result of suffering from loss of appetite and reduced sun exposure. Previous research showed that the levels of serum calcium and 25(OH)D3 were closely related to clinical prognosis of breast cancer patients with bone metastases following treatment with zoledronic acid, but the predictive value of serum calcium and 25(OH)D3 in patients with lung cancer metastatic to the skeleton has not been published in the literature. The aim of this study is to evaluate the relevance and meaning of calcium metabolism for skeletal-related events and overall survival in lung cancer patients with bone metastases undergoing treatment with zoledronic acid by analyzing the changes of level of serum calcium and 25(OH)D3.Methods and Materials1. This was an observational and prospective study which including 86 cases of lung cancer with bone metastases,60 cases of lung cancer without bone metastases and 57 normal controls between January 2013 and June 2014.2. All lung cancer patients with bone metastases were treated with ZA (4 mg every 4 weeks for at least 6 months). Serum levels of calcium and 25(OH)D3 were measured at baseline among cases with and without bone metastases and normal controls, and monitor of dynamic changes of serum calcium and 25(OH)D3 at 2,4,6, 8,10 and 12 months after the beginning of treatment.3. Data on SREs development and overall survival of lung cancer patients with bone metastases were recorded, and the correlation between calcium metabolism and the clinical significance of prognosis in lung cancer patients with bone metastases were analyzed.Results1. The baseline level of serum calcium in cases with bone metastases was significantly higher than cases without bone metastases and normal controls(2.91 ±0.17 mmol/L vs 2.55±0.12 mmol/L, P<0.05; 2.91±0.17 mmol/L vs 2.43±0.20 mmol/L, P<0.05), but the level of 25(OH)D3 was lower than other two groups(39.65±9.57 ng/mL vs 45.88±7.24 ng/mL, P<0.05; 39.65±9.57 ng/mL vs 57.82±8.93 ng/mL,P<0.05).2. After receiving the treatment with ZA, the level of serum calcium significantly decreased comparing with baseline (2.61±0.12 mmol/L vs 2.91±0.17 mmol/L, P<0.05), but the level of 25(OH)D3 had few differences with baseline(41.02±7.54 ng/mL vs39.65±9.57 ng/mL,P>0.05).3. Multivariate Logistic regression analysis showed elevated calcium and declined 25(OH)D3 were independent risk factors of SREs (OR=4.566, P=0.014; OR=3.843,P=0.040).4. The median time to SREs in patients with elevated calcium was significantly shorter than that in patients with declined calcium (10.0 vs 12.0 months, P<0.05), but the median survival time(MST) in two groups had no difference (13.5 vs 16.0 months, P>0.05). The median time to SREs and MST in patients with declined 25(OH)D3 was significantly shorter than those in patients with elevated 25(OH)D3 (9.0 vs 13.0 months, P<0.05; 12.5 vs 18.0months, P<0.05).Conclusion1. Serum calcium and 25(OH)D3 can be considered suitable predictors for SREs in lung cancer patients with bone metastases treated with ZA.2. Serum 25(OH)D3 is also appropriate to evaluate the overall prognosis in lung cancer patients with bone metastases.PART TWO The expression of calcyphosine in bone metastases of non-small cell lung cancer and correlation studyBackground and purposeLung cancer is one of the most common malignancies and the leading cause of cancer death worldwide. Non-small-cell-lung cancer(NSCLC) is the most common subtype of lung cancer, which accounts for approximately 70% of lung cancer diagnoses. During the course of the disease, almost a third of lung cancer patients have distant tumor spread when first diagnosed, and bone metastases occur most frequently in advanced lung cancer patients. Underlying pathophysiology involves reciprocal interactions between tumor cells and the bone microenvironment that may provoke disruption of the balanced regulation of the processes of bone formation, mediated by osteoblasts, and bone resorption, mediated by osteoclasts. The discharge of calcium from bone tissue is promoted by cytokines secretion of tumor cells, in the first part of this paper, we fond the level of serum calcium was associated with bone metastases of lung cancer, meanwhile calcium plays a very important role as an important intracellular secondary messenger in bone metastases of cancer, and earlier studies showed that calcium-binding proteins, the physiological form of calcium, could execute the regulatory biological function in a number of malignant cells. A recent research suggests that the extent of calcyphosine(CAPSl) expression, as the newly-found calcium-binding protein, is closely correlated with the prognosis in patients with endometrial carcinoma. In previous study of our laboratory, we had discovered that CAPS1 was selectively over-expressed in NSCLC bone metastases tissue by proteomic techniques, so we select CAPS1 and study its expression and potential effect in bone metastases of NSCLC.Methods and Materials1. To detect the expression of CAPS1 in bone metastases of NSCLC, the total RNAs and proteins of NSCLC bone metastases tissue confirmed by pathological evidence, primary malignant bone tumor(osteosarcoma) and normal bone tissue were collected and examined by real-time PCR and western blot assays.2. Real-time PCR and western blot assays were used to demonstrate the protein and RNA expression of CAPS 1 in NSCLC cell lines with different capability of bone metastases.3. A lentivirus vector was successfully constructed to increase the expression of CAPS1 in NSCLC cell, and the interfering efficiency was confirmed by Real-time PCR and Western blot assays.4. To investigate effects of CAPS1 on bone metastases in NSCLC cell in vitro, CCK-8 and flow cytometry techniques were applied to detect the proliferative capability, apoptosis and cell cycle distribution, meanwhile the effects of CAPS 1 on the invasion and migration of NSCLC cell were assayed with wound healing assay and transwell methods.Results1. CAPS1 showed specific and high expression in NSCLC bone metastases.2. CAPS1 expression in none-bone-metastatic NSCLC cell line A549 was significantly lower than that in bone-metastatic NSCLC cell line H1299, H460 and SPC-A1, it indicates that CAPS1 maybe associated with bone metastases in NSCLC.3. The NSCLC cell line A549 increasing the expression of CAPS1 was successfully constructed.4. Over-expression of CAPS1 in NSCLC cell line A549 demonstrated higher proliferative ability, stronger invasiveness and more aggressive metastatic potential than normal-expression of CAPS1 in A549.ConclusionCAPS1 is closely related to bone metastases of NSCLC, over-expression of CAPS1 can significantly enhance the proliferative ability, invasion and metastasis potential of NSCLC cell in vitro. |
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