Abstract
Purpose
Vocal fold leukoplakia is a premalignant precursor of squamous cell carcinoma. Although many efforts have been contributed to therapy of this disease, none exhibits a satisfactory result. The aims of this study were to investigate the effectiveness and feasibility of andrographolide therapy in vocal fold leukoplakia and to explore the preliminary mechanism underlying.
Materials and Methods
Forty-one eligible patients were enrolled in the study. The patients were treated for 10-minute exposures of 5 ml (25 mg/ml) andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Electronic laryngoscope was used to observe the condition of vocal fold leukoplakia during the treatment. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months. Toxic reactions of treatments were evaluated on the basis of the standards of the United States MD Anderson Cancer Center. Moreover, laryngeal carcinoma cell line Hep2 was applied to explore the mechanism of effect of andrographolide. Anti-proliferative effect on Hep2, cell nuclear morphology, express of mitogen-activated protein kinases (MAPK) and pro-apoptotic protein were detected after andrographolide treatment.
Results
We found that andrographolide exhibited significant curative effects on treatments, which were accompanied by thinning of the lesion of leukoplakia, reduction in the whitish surface area, and return of pink or red epithelium. A complete response up to 85% was observed, and no toxic side effect events occurred during the study. No patient with a complete response had a recurrence in the follow-up. Moreover, cellular experiments in Hep2 indicated that andrographolide activated MAPK pathway and caspase cascade, and finally induced apoptosis in laryngeal carcinoma cell.
Conclusions
The advantages of andrographolide are connected with minimally invasive and localized character of the treatment and no damage of collagenous tissue structures, which are more convenient and less painful for patients. These results suggest that andrographolide treatment is a viable strategy for curing vocal fold leukoplakia.
1
Introduction
Vocal fold leukoplakia is the clinical term used to describe a white patch on the mucosa of vocal cords or larynx, which is related to tobacco smoking, excessive drinking or voice abuse . Pathology of biopsy indicates that leukoplakia is attributed to epithelial dysplasia and hyperkeratosis . Moreover, hyperkeratosis is considered as an important morphological feature in the process of cancerogenesis. It is reported that the presence of epithelial dysplasia can increase the risk of malignant transformation by up to 40% . Although many efforts, such as surgery and laser ablation, have been contributed to treat the disease, results are still not satisfactory. Disadvantages of common treatments are revealed, including adverse effects, mucosa lesions and recurrences . Therefore, studies on improvement in therapy of vocal fold leukoplakia are urgently needed.
Andrographolide ( Fig. 1 ), a labdane diterpenoid, is the most active and important constituent of the medicinal plant Andrographis paniculata , which has long been used as a herbal medicine to prevent and treat upper respiratory tract infections, diarrhea, rheumatoid arthritis, and inflammation in Asia, Scandinavia and America . It is reported that andrographolide could suppress IL-2 production and T-cell proliferation in a mixed lymphocyte reaction , and inhibit the activation of NF-кB in asthma . Recently, andrographolide shows activities in anticancer , including inhibition of cell growth of hepatocellular carcinoma , cervical carcinoma , prostatic adenocarcinoma , and breast carcinoma . Meanwhile, more and more studies demonstrate that andrographolide has advantages of extensive pharmacological effects and less side effects in therapy. However, the curative efficacy of andrographis in vocal fold leukoplakia has not been investigated so far, and the mechanism is still not clear.
It is known that aerosol inhalation is a direct method of drug administration for treatment of respiratory diseases. The great advantage lies in the fact that the drug deposited from inhalation results in a high concentration at the site of action . In addition, a much lower concentration is produced in the systemic circulation following absorption, and it will reduce the possibility of adverse reactions.
In the present study, andrographolide-inhaled atomization was applied to cure vocal fold leukoplakia in the clinic, and laryngeal carcinoma cell line Hep2 was used as an in vitro model system to explore the mechanism underlying of andrographolide. Moreover, therapeutic toxicity was under strict monitor in order to evaluate whether andrographolide could be the candidate as a preventive agent for vocal fold leukoplakia.
2
Materials and methods
2.1
Patients
The study was performed at the Department of Otolaryngological Surgery of First People’s Hospital Affiliated to Huzhou University. The study protocol complied with the Declaration of Helsinki and obtained approval from Ethical Committees. Candidates were identified in the head and neck oncology clinic. Patients, median age 46 years (range, 32–65 years), with at least one grossly visible leukoplakia lesion, with or without dysplasia, measuring 10 mm or more in diameter were included. Finally, 41 patients were enrolled in the study between January 2010 and June 2012. The detailed and important characteristics of patients were shown in Table 1 .
| Characteristics | Number of patients |
|---|---|
| Sex | |
| Male | 34 |
| Female | 7 |
| Histology | |
| Atypical hyperplasia | 10 |
| Moderate dysplasia | 25 |
| Severe dysplasia | 3 |
| Hyperkeratosis | 3 |
| History of risk factors | |
| Tobacco and alcohol | 12 |
| Tobacco only | 9 |
| Alcohol only | 5 |
| None identified | 15 |
2.2
Drugs and chemicals
Andrographolide injection (each 5-ml vial contains active components equivalent to 125 mg of andrographolide) was purchased from Qing Feng Pharmaceutical Co., Ltd (Ganzhou, China), whose products met the commercial quality control according to the China Pharmacopoeia 2010. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) was purchased from Sigma Chemical Company (St Louis, MO). MTT was dissolved in RPMI 1640 to make a 5-mg/ml solution. Antibodies for p-JNK, p-p38, cleaved caspase-3 were purchased from Cell Signal Technology Inc. (Danvers, MA) and JNK, p38, procaspase-3, PARP, Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary anti-mouse, anti-goat and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology. The western blot detection reagent ECL was purchased from Pierce Biotechnology (Rockford, IL).
2.3
Andrographolide treatment and follow-up
Atomization device (PARI BOY N, PARI, Germany) was applied for aerosols generation according to the manufacturer’s protocol. Briefly, air was directed into the baffle and adjusted to a flow rate of 3.0 L/min. A transducer in the device was used to generate the initial droplets of an andrographolide solution, which were subsequently dried by a silica drying column. Then aerosols were generated with the higher-frequency transducers.
The patients were treated for 10-minute exposures of 5-ml andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months.
2.4
Evaluation of laryngoscope
Electronic laryngoscope (PCH03-D, XiON GmbH, Germany) was applied to observe the condition of vocal fold leukoplakia, and excised the leukoplakia only a little for histopathological analysis.
2.5
Histopathological analysis
Vocal fold leukoplakia samples were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin for histopathological analysis. The sections of vocal fold leukoplakia were examined under light microscopy.
2.6
Evaluation of voice quality
The perceptive assessment of the voice was performed according to the Sittel’s scale after treatment . A trained otolaryngologist rated each voice independently for communication ability in a grade from poor to normal.
2.7
Evaluation of toxic reactions
Patients were evaluated for toxic reactions every 2 weeks during the study and at 3-month intervals during follow-up. Toxicity was evaluated on the basis of subjective and objective symptoms. The criteria for grading toxicity were based on the standards of the United States MD Anderson Cancer Center , which include the common toxicity criteria of the US National Cancer Institute and other centers. Grade I toxicity was mild (i.e., cheilitis easily controlled with emollients); grade II, moderate and tolerable (pruritus causing discomfort or generalized mild dermatitis); grade III, severe and intolerable (including poorly controlled conjunctivitis requiring artificial tears); and grade IV, most severe and life-threatening (generalized exfoliative dermatitis with or without systemic infection).
2.8
MTT assay
Hep2 cells were seeded in 96-well plates (6 × 10 3 /well) for 24 h, and subsequently treated with different concentrations of andrographolide for 48 h. Viable cells were determined using MTT assay. MTT was added (10.0 μl/well), and plates were incubated for a further 4 h at 37 °C. The purple formazan crystals were dissolved in 100 μl DMSO. After the crystal dissolved, the plates were read on an automated microplate spectrophotometer (Thermo Multiskan Spectrum, Thermo Electron Corporation, Waltham, MA) at 570 nm. The concentration of drug inhibition for 50% of cells (IC50) was calculated using the PrismPad computer program (GraphPad Software Inc.) with Microcomputers.
2.9
DAPI staining assay
Hep2 cells were cultured in 24-well plates and treated with andrographolide for 48 h. Washed the cells twice with PBS and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) which diluted with 0.1% Triton X-100 for 5 min. Observed the changes of nuclei with fluorescence microscope (DMI 4000 B, Leica, Germany). Cells with chromatin condensation and nuclear fragmentation were considered apoptotic.
2.10
Western blot analysis
The protein samples of cells were extracted in lysate buffer which consist of 50.0 mM NaCl, 50.0 mM Tris–HCl, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 0.1% SDS. Total protein concentration of whole cell lysates was determined using BioRad BCA method (Pierce, Rockford, IL). Total protein of 40.0–60.0 μg of was loaded per lane and fractionated on 10%–15% Tris–glycine precast gels, transferred to PVDF membrane (Millipore, Bedford, MA). The membranes were blocked with 5% non-fat dry milk in 0.01 M Tris-buffered saline (TBS) (pH 7.4) and 0.1% Tween-20 (TBST) at room temperature for 2 h. Subsequently, the membrane was incubated with primary antibodies directed against target proteins overnight at 4 °C. The final dilutions for primary antibodies were as follows: JNK, p38, procaspase-3, 1: 500; p-JNK, p-p38, cleaved caspase-3, PARP, and Actin, 1: 1000. After the membranes were washed, they were incubated for 1 h with HRP-labeled secondary antibodies diluted at 1: 5000 in TBST. Proteins were visualized using ECL.
2.11
Statistical analysis
The significance of differences between the values of the groups was determined with the one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc test using SPSS 16.0 software. P < 0.05 was accepted as statistically significant.
2
Materials and methods
2.1
Patients
The study was performed at the Department of Otolaryngological Surgery of First People’s Hospital Affiliated to Huzhou University. The study protocol complied with the Declaration of Helsinki and obtained approval from Ethical Committees. Candidates were identified in the head and neck oncology clinic. Patients, median age 46 years (range, 32–65 years), with at least one grossly visible leukoplakia lesion, with or without dysplasia, measuring 10 mm or more in diameter were included. Finally, 41 patients were enrolled in the study between January 2010 and June 2012. The detailed and important characteristics of patients were shown in Table 1 .
| Characteristics | Number of patients |
|---|---|
| Sex | |
| Male | 34 |
| Female | 7 |
| Histology | |
| Atypical hyperplasia | 10 |
| Moderate dysplasia | 25 |
| Severe dysplasia | 3 |
| Hyperkeratosis | 3 |
| History of risk factors | |
| Tobacco and alcohol | 12 |
| Tobacco only | 9 |
| Alcohol only | 5 |
| None identified | 15 |
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