In Situ Electrodeposited Synthesis of Electrochemiluminescent Ag Nanoclusters as Signal Probe for Ultrasensitive Detection of Cyclin-D1 from Cancer Cells.

Metal nanoclusters (NCs) as a new type of electrochemiluminescence (ECL) nanomaterials have attracted great attention, but their applications are limited due to relatively low luminescent efficiency and a complex preparation process. Herein, an ultrasensitive ECL biosensor for the detection of Cyclin-D1 (CCND1) was designed by utilizing in situ electrogenerated silver nanoclusters (AgNCs) as ECL emitters and Fe3O4-CeO2 nanocomposites as a coreaction accelerator. The ECL luminous efficiency of AgNCs on the electrode could be significantly enhanced with the use of the Fe3O4-CeO2 for accelerating the reduction of S2O82- to generate the strong oxidizing intermediate radical SO4•-. As a result, the assay for CCND1 detection achieved excellent sensitivity with a linear range from 50 fg/mL to 50 ng/mL and limit of detection down to 28 fg/mL. Impressively, the efficiency of Traditional Chinese Medicines (TCM), sophorae, toward MCF-7 cells was successfully investigated due to the overexpression of CCND1 in relation to the growth and metastasis of MCF-7 human breast cancer cells. In general, the proposed strategy provided an effective method for anticancer drug screening and expanded the application of metal NCs in ultrasensitive biodetection.

Cyclin D1 as a therapeutic target of renal cell carcinoma- a combined transcriptomics, tissue microarray and molecular docking study from the Kingdom of Saudi Arabia.

BACKGROUND: Renal cell carcinoma (RCC) is a seventh ranked malignancy with poor prognosis. RCC is lethal at metastatic stage as it does not respond to conventional systemic treatments, and there is an urgent need to find out promising novel biomarkers for effective treatment. The goal of this study was to evaluate the biomarkers that can be potential therapeutic target and predict effective inhibitors to treat the metastatic stage of RCC. METHODS: We conducted transcriptomic profiling to identify differentially expressed genes associated with RCC. Molecular pathway analysis was done to identify the canonical pathways and their role in RCC. Tissue microarrays (TMA) based immunohistochemical stains were used to validate the protein expression of cyclinD1 (CCND1) and were scored semi-quantitatively from 0 to 3+ on the basis of absence or presence of staining intensity in the tumor cell. Statistical analysis determined the association of CCND1 expression with RCC. Molecular docking analyses were performed to check the potential of two natural inhibitors, rutin and curcumin to bind CCND1. RESULTS: We detected 1490 significantly expressed genes (1034, upregulated and 456, downregulated) in RCC using cutoff fold change 2 and p value < 0.05. Hes-related family bHLH transcription factor with YRPW motif 1 (HEY1), neuropilin 2 (NRP2), lymphoid enhancer-binding factor 1 (LEF1), and histone cluster 1 H3h (HIST1H3H) were most upregulated while aldolase B, fructose-bisphosphate (ALDOB), solute carrier family 12 (SLC12A1), calbindin 1 (CALB1) were the most down regulated genes in our dataset. Functional analysis revealed Wnt/ß-catenin signaling as the significantly activated canonical pathway (z score = 2.53) involving cyclin D1 (CCND1). CCND1 was overexpressed in transcriptomic studies (FC = 2.26, p value = 0.0047) and TMA results also showed the positive expression of CCND1 in 53 % (73/139) of RCC cases. The ligands - rutin and curcumin bounded with CCND1 with good affinity. CONCLUSION: CCND1 was one of the important upregulated gene identified in microarray and validated by TMA. Docking study showed that CCND1 may act as a potential therapeutic target and its inhibition could focus on the migratory, invasive, and metastatic potential of RCC. Further in vivo and in vitro molecular studies are needed to investigate the therapeutic target potential of CCND1 for RCC treatment.

Shenhua Tablet inhibits mesangial cell proliferation in rats with chronic anti-Thy-1 nephritis.

BACKGROUND: In China, mesangial proliferative glomerulonephritis (MsPGN) is one of the most common kidney diseases. In this study, we treated a rat model of chronic anti-Thy-1 MsPGN with Shenhua Tablet and evaluated whether the tablet was able to protect the kidney function. Thirty-six Wistar rats were randomly divided into six groups: (1) Sham surgery (Sham); (2) anti-Thy-1 nephritis model (Thy-1); (3) anti-Thy-1 nephritis model + irbesartan-treated (Irb); (4) anti-Thy-1 nephritis model + low-dose of Shenhua Tablet (SHL); (5) anti-Thy-1 nephritis model + medium-dose of Shenhua Tablet (SHM); (6) anti-Thy-1 nephritis model + high-dose of Shenhua Tablet (SHH). RESULTS: Thirteen weeks after drug treatment, urinary proteins were quantified and renal pathological changes were thoroughly examined at the time point of 24 h. Meanwhile, the expression levels of p-Erk1/2, cyclin D1 and p21 at the renal cortex were also tested. The levels of urinary proteins and total cholesterol in the blood were significantly reduced in rats treated with any drug tested in this study. The level of triglyceride was significantly reduced in all three Shenhua Tablet-treated groups. Renal pathomorphological scores were significantly improved in groups of Irb, SHM and SHH. Mesangial cell proliferation was significantly inhibited in any drug-treated group. p-Erk1/2 and cyclin D1 were downregulated whereas p21 was upregulated in the renal cortex. CONCLUSIONS: Our study indicated that Shenhua Tablet is able to inhibit the abnormal proliferation of mesangial cells and to prevent kidney damage, which is likely associated with downregulation of p-Erk1/2 and reduced activity of its downstream target-cyclin D1.

Shenhua Tablet inhibits mesangial cell proliferation in rats with chronic anti-Thy-1 nephritis

BACKGROUND: In China, mesangial proliferative glomerulonephritis (MsPGN) is one of the most common kidney diseases. In this study, we treated a rat model of chronic anti-Thy-1 MsPGN with Shenhua Tablet and evaluated whether the tablet was able to protect the kidney function. Thirty-six Wistar rats were randomly divided into six groups: (1) Sham surgery (Sham); (2) anti-Thy-1 nephritis model (Thy-1); (3) anti-Thy-1 nephritis model + irbesartan-treated (Irb); (4) anti-Thy-1 nephritis model + low-dose of Shenhua Tablet (SHL); (5) anti-Thy-1 nephritis model + medium-dose of Shenhua Tablet (SHM); (6) anti-Thy-1 nephritis model + high-dose of Shenhua Tablet (SHH). RESULTS: Thirteen weeks after drug treatment, urinary proteins were quantified and renal pathological changes were thoroughly examined at the time point of 24 h. Meanwhile, the expression levels of p-Erk1/2, cyclin D1 and p21 at the renal cortex were also tested. The levels of urinary proteins and total cholesterol in the blood were significantly reduced in rats treated with any drug tested in this study. The level of triglyceride was significantly reduced in all three Shenhua Tablet-treated groups. Renal pathomorphological scores were significantly improved in groups of Irb, SHM and SHH. Mesangial cell proliferation was significantly inhibited in any drug-treated group. p-Erk1/2 and cyclin D1 were downregulated whereas p21 was upregulated in the renal cortex. CONCLUSIONS: Our study indicated that Shenhua Tablet is able to inhibit the abnormal proliferation of mesangial cells and to prevent kidney damage, which is likely associated with downregulation of p-Erk1/2 and reduced activity of its downstream target-cyclin D1.

Simultaneous expression analysis of vitamin D receptor, calcium-sensing receptor, cyclin D1, and PTH in symptomatic primary hyperparathyroidism in Asian Indians.

BACKGROUND: To explore underlying molecular mechanisms in the pathogenesis of symptomatic sporadic primary hyperparathyroidism (PHPT). MATERIALS AND METHODS: Forty-one parathyroid adenomas from patients with symptomatic PHPT and ten normal parathyroid glands either from patients with PHPT (n=3) or from euthyroid patients without PHPT during thyroid surgery (n=7) were analyzed for vitamin D receptor (VDR), calcium-sensing receptor (CASR), cyclin D1 (CD1), and parathyroid hormone (PTH) expressions. The protein expressions were assessed semiquantitatively by immunohistochemistry, based on percentage of positive cells and staining intensity, and confirmed by quantitative real-time PCR. RESULTS: Immunohistochemistry revealed significant reductions in VDR (both nuclear and cytoplasmic) and CASR expressions and significant increases in CD1 and PTH expressions in adenomatous compared with normal parathyroid tissue. Consistent with immunohistochemistry findings, both VDR and CASR mRNAs were reduced by 0.36- and 0.45-fold change (P<0.001) and CD1 and PTH mRNAs were increased by 9.4- and 17.4-fold change respectively (P<0.001) in adenomatous parathyroid tissue. PTH mRNA correlated with plasma PTH (r=0.864; P<0.001), but not with adenoma weight, while CD1 mRNA correlated with adenoma weight (r=0.715; P<0.001). There were no correlations between VDR and CASR mRNA levels and serum Ca, plasma intact PTH, or 25-hydroxyvitamin D levels. In addition, there was no relationship between the decreases in VDR and CASR mRNA expressions and the increases in PTH and CD1 mRNA expressions. CONCLUSIONS: The expression of both VDR and CASR are reduced in symptomatic PHPT in Asian Indians. In addition, CD1 expression was greatly increased and correlated with adenoma weight, implying a potential role for CD1 in adenoma growth and differential clinical expression of PHPT.

Unconventional role of the inwardly rectifying potassium channel Kir2.2 as a constitutive activator of RelA in cancer.

The constitutive activation of NF-κB is a major event leading to the initiation, development, and progression of cancer. Recently, we showed that the size of preestablished tumors was reduced after the depletion of Kir2.2, an inwardly rectifying potassium channel. To determine the precise mechanism of action of Kir2.2 in the control of tumor growth, we searched for interacting proteins. Notably, NF-κB p65/RelA was identified as a binding partner of Kir2.2 in a yeast two-hybrid analysis. Further analyses revealed that Kir2.2 directly interacted with RelA in vitro and coimmunoprecipitated with RelA from cell lysates. Kir2.2 increased RelA phosphorylation at S536 and facilitated its translocation from the cytoplasm to the nucleus, thereby activating the transcription factor and increasing the expression level of NF-κB targets, including cyclin D1, matrix metalloproteinase (MMP)9, and VEGF. Kir2.2 was overexpressed in human cancer and the expression level was correlated with increased colony formation and tumor growth in mouse tumor models. On the basis of these findings, we propose an unconventional role for Kir2.2 as a constitutive RelA-activating protein, which is likely to contribute to tumor progression in vivo.

Effects of beta-carotene supplementation on molecular markers of lung carcinogenesis in male smokers.

Two primary prevention trials unexpectedly showed adverse effects of supplemental beta-carotene on lung cancer incidence in cigarette smokers. To elucidate the molecular mechanisms that might underlie these effects, we studied the immunohistochemical expression of cytochrome P450 1A1, 1A2, and 2E1, retinoic acid receptor beta, activated protein-1 elements, cyclin D1, and Ki67 in lung tumors and, when available, adjacent normal tissues obtained from incident cases in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Archival lung tissue was available from 52 men randomized to receive 20 mg of beta-carotene per day and 30 men randomized to the placebo arm, all of whom were diagnosed with incident non-small-cell lung carcinoma during the course of the trial and subsequently underwent radical pulmonary resection. In normal-appearing bronchial epithelium, positive staining for cyclin D1 was observed in 23% of cases in the beta-carotene group and 0% of cases in the placebo group (based on only 3 of 13 versus 0 of 11 cases staining positively, however; P = 0.04), with no differences in expression noted in lung tumor tissue (P = 0.48). There were no statistically significant differences in Ki67 expression in normal or cancerous lung tissue between intervention groups, although a small increase in staining in tumors was noted among cases in the beta-carotene versus placebo group (88% versus 71% of cases stained positive, respectively; P = 0.13). Contrary to expectation, beta-carotene supplementation had no apparent effect on retinoic acid receptor-beta expression. These findings suggest that male smokers supplemented with beta-carotene may have had an increased risk of lung cancer due to aberrant cell growth, although our results are based on a relatively small number of cases and require confirmation in other completed trials of beta-carotene supplementation.

Akt/cAMP-responsive element binding protein/cyclin D1 network: a novel target for prostate cancer inhibition in transgenic adenocarcinoma of mouse prostate model mediated by Nexrutine, a Phellodendron amurense bark extract.

PURPOSE: Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)-mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protect TRAMP mice from developing prostate cancer. EXPERIMENTAL DESIGN: Eight-week-old TRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules. RESULTS: We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylation-defective CREB inhibited cyclin D1 transcriptional activity. CONCLUSIONS: The current study shows that Nexrutine-mediated targeting of Akt/CREB-induced activation of cyclin D1 prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.

Anti-proliferative effects of green tea polyphenol EGCG on Ha-Ras-induced transformation of intestinal epithelial cells.

Oncogenic Ras mutations are frequently observed in colorectal cancer and participate in neoplastic transformation of intestinal epithelial cells. Accumulating evidence demonstrates the chemopreventive properties of green tea on colon carcinogenesis. Here we investigated the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), to inhibit proliferation of intestinal epithelial cells (RIE-1) transfected with an inducible Ha-Ras(Val12) cDNA. EGCG inhibited cell proliferation induced by oncogenic Ras and blocked cell cycle transition at G1 phase via inhibition of cyclin D1 expression. The EGCG IC(50) was 42microM in transformed cells and 81microM in non-transformed cells. EGCG also promoted E-cadherin expression, which is downregulated by Ras transformation. This study demonstrates the potential of the natural compound EGCG as an effective adjuvant therapy for colon tumors bearing Ras mutations.

Dietary supplementation with silymarin inhibits 3,2'-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in male F344 rats.

PURPOSE: Silymarin has been shown to be a potent anticarcinogenic agent. Here, we investigated the modifying effects of dietary feeding with a naturally occurring polyphenolic antioxidant flavonoid silymarin on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis in male F344 rats. EXPERIMENTAL DESIGN: Male F344 rats were given s.c. injections of DMAB (25 mg/kg body weight) every other week for 20 weeks. They also received the experimental diet containing 100 or 500 ppm silymarin for 40 weeks, starting 1 week after the last dosing of DMAB. All of the rats were sacrificed 60 weeks after the start of the experiment. Histopathology and immunohistochemistry for proliferative cell nuclear antigen, cyclin D1, and apoptotic indices were done in the prostatic lesions, including invasive adenocarcinomas, intraepithelial neoplasms, and nonlesional glands. RESULTS: Dietary feeding with 500 ppm silymarin significantly inhibited the incidence of prostatic adenocarcinoma when compared with the DMAB-alone group (17.6% versus 50.0%, P < 0.05). The proliferative cell nuclear antigen- and cyclin D1-positive indices in adenocarcinomas, prostatic intraepithelial neoplasm, and nonlesional glands in rats treated with DMAB and silymarin were slightly lower than that of the DMAB-alone group. Also, dietary administration of silymarin increased apoptotic index in prostatic adenocarcinoma by measuring immunohistochemically positive nuclei for ssDNA. CONCLUSIONS: Our results indicate that silymarin exerts chemopreventive ability against chemically induced prostatic carcinogenesis through apoptosis induction and modification of cell proliferation.

Dietary retinoic acid supplementation stimulates intestinal tumour formation and growth in multiple intestinal neoplasia (Min)/+ mice.

Chemopreventive activity by retinoic acid (RA) has been demonstrated previously in rat colon. The spontaneous tumourigenesis in the Min/+ mouse, which harbours a germline mutation in the tumour suppressor gene adenomatous polyposis coli (Apc), is characterized by inactivation of Apc, nuclear accumulation of beta-catenin and the enhanced expression of specific genes activated by T cell factor (TCF)/beta-catenin signalling. Recently it was reported that beta-catenin interacts with retinoic acid receptor in a retinoid-dependent manner, reducing beta-catenin/TCF regulated transcription. Our hypothesis was therefore that dietary supplementation with all-trans RA may inhibit the Apc-driven tumourigenesis in Min/+ mice. Surprisingly, in two different experiments the results showed that dietary RA significantly stimulated both the formation and growth of small intestinal tumours. In the first experiment Min/+ mice were exposed to 50 mg 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine/kg bodyweight at day 3-6 after birth and then treated with 50 mg/kg dietary RA in 1-3 weeks from the age of 2 weeks. In the second experiment the mice were not treated with carcinogen, and the diet was supplemented with 5 or 10 mg/kg RA from the age of 4 weeks until termination of the experiment at 11 weeks. Immunohistochemical studies revealed no differences in beta-catenin, cyclin D1 or proliferating cell nuclear antigen staining following RA treatment. There was no intestinal toxicity in mice fed 10 mg/kg RA, indicating that the increased tumourigenesis in Min/+ mice is a specific effect of all-trans RA.

Effects of hypothalamic neuropeptides on extracellular signal-regulated kinase (ERK1 and ERK2) cascade in human tumoral pituitary cells.

The G protein-coupled receptor (GPCR) activation has been demonstrated to affect the ERK1/2 cascade in different cell lines. We investigated the effects of hypothalamic neuropeptides acting via GPCR on this pathway in GH-secreting (GH-oma) and nonsecreting (NFPA) pituitary adenomas. GHRH increased ERK1/2 activity (236 +/- 80%) in both gsp- and gsp+ GH-omas, this effect being almost completely abolished by protein kinase C (PKC) blockade. Both GnRH and pituitary adenylate-activating peptide caused a similar PKC-dependent activation of ERK1/2 in most NFPA. Increasing cAMP by forskolin caused a protein kinase A-dependent increase of ERK activity (287 +/- 37%) in GH-omas and had no effect in NFPA. ERK cascade blockade in GH-omas did not affect basal and GHRH-stimulated GH release, whereas it totally prevented the 3-fold increase in cyclin D1 protein expression induced by GHRH. In conclusion, this study demonstrated that in pituitary adenomas the activation of GPCR by neurohormones caused a PKC-dependent activation of ERK1/2 cascade that, at least in GH-omas, resulted to be involved in cyclin D1 induction by GHRH. Moreover, a stimulatory effect of the protein kinase A-dependent pathway on ERK1/2 cascade occurred selectively in GH-omas, probably contributing to the mitogenic potential of the cAMP pathway in this cell type.

Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver.

The effects of tea polyphenols and tea pigments on rat liver precancerous lesions and some cell cycle regulators were studied. A modified Solt-Farber model in rats was established by multiple low-dosage of N-nitrosodiethylamine (NDEA) i.p. injections, followed by i.p. CCl(4) injection and partial hepatectomy. Sixty male Wistar rats were randomly divided into four groups: positive control group, two tea-treated groups, and negative control group. Rats in tea-treated groups were given tea polyphenols (0.1%) and tea pigments (0.1%) in drinking fluid during the whole experiment. The number and area of glutathione S-transferase P (GST-P)-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western blotting assay was carried out to detect the expression of cyclin D1, CDK4, and P21(WAF1/CIP1) on whole liver extract. At the end of the experiment (56 days), the number and area of GST-P-positive foci in liver increased significantly in carcinogen-administered positive control group, whereas no GST-P-positive foci were found in the negative control group in which animals did not receive carcinogen exposure. The number and area of GST-P-positive foci in tea-treated, carcinogen-exposed groups were significantly reduced as compared with the positive control group. It was also found that the expression of P21(WAF1/CIP1) was significantly induced and the expression of cyclin D1 and CDK4 was significantly inhibited in tea-treated groups. These results suggest that tea polyphenols and tea pigments are effective in preventing the precancerous liver lesions in rats, and modulation of cell cycle by regulating cell cycle regulators may be a possible mechanism.

Suppression of human pancreatic carcinoma cell growth and invasion by epigallocatechin-3-gallate.

INTRODUCTION: The consumption of green tea is associated with a lower risk of several types of human carcinomas. A number of studies have focused on the possible mechanisms of cancer prevention by tea extracts, especially polyphenols such as epigallocatechin-3-gallate (EGCG). AIMS AND METHODOLOGY: Green tea-derived EGCG was tested in human pancreatic carcinoma cells. The cells (PANC-1, MIA PaCa-2, and BxPC-3) were treated with different doses of EGCG (0, 25, 50, 100, and 200 micromol/L) for 48 hours in culture medium. Proliferation of pancreatic carcinoma cells was measured by means of the WST-1 colorimetric assay. For the study of cell invasion, the cells were incubated with 100 micromol/L EGCG for 2 hours. Then, the cells were added into the cell insert, coated with Matrigel basement membrane matrix. After incubation at 37 degrees C for 24 hours, the cells that had invaded through the Matrigel were counted visually under the microscope. RESULTS: The growth of all three pancreatic carcinoma cells was significantly suppressed by EGCG treatment in a dose-dependent manner. EGCG treatment caused significant suppression of the invasive ability of pancreatic carcinoma cells PANC-1, MIA PaCa-2, and BxPC-3 but did not affect the cell cycle protein cyclin D1. CONCLUSION: EGCG may be a potent biologic inhibitor of human pancreatic carcinomas, reducing their proliferative and invasive activities.

Effects of physiological versus pharmacological beta-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets.

There remains a remarkable discordance between the results of observational epidemiological studies and intervention trials using beta-carotene as a potential chemopreventive agent. One question that needs to be examined is whether the adverse outcomes of human beta-carotene trials are related to the large doses of beta-carotene that were administered. In the present study, ferrets were given a physiological (low) dose or a pharmacological (high) dose of beta-carotene supplementation (0.43 mg versus 2.4 mg/kg body wt/day, which is equivalent to 6 mg versus 30 mg/day in humans) and exposed to cigarette smoke for 6 months. We investigated the effects of these doses of beta-carotene on retinoid concentrations, expression of retinoic acid receptors (RARs), activator protein 1 (AP-1; c-Jun and c-Fos), cyclin D1, proliferating cellular nuclear antigen (PCNA), and histopathological changes in the lungs of both normal and cigarette smoke-exposed ferrets. Thirty-six male ferrets were treated in six groups-control, smoke-exposed (SM), low-dose beta-carotene (LBC), high-dose beta-carotene (HBC), low-dose beta-carotene plus smoke exposure (LBC+SM) or high-dose beta-carotene plus smoke exposure (HBC+SM)-for 6 months. Retinoic acid concentration and RAR beta gene expression, but not expression of RAR alpha and RAR gamma, was reduced in the lung tissue of HBC+SM, HBC, SM and LBC+SM ferrets, but not in that of LBC ferrets, as compared with the control group. Expression of AP-1 and PCNA was greater in HBC+SM, HBC, SM and LBC+SM ferrets, but not in the LBC ferrets, as compared with the control group. Increased amounts of cyclin D1 and keratinized squamous metaplasia were observed in the lung tissue of HBC+SM, HBC and SM groups but not in that of the LBC+SM, LBC or control groups. These data suggest that, in contrast with a pharmacological dose of beta-carotene, a physiological dose of beta-carotene in smoke-exposed ferrets has no potentially detrimental effects and may afford weak protection against lung damage induced by cigarette smoke.