Enhancement of treatment efficacy of hepatic tumours using Trans-arterial-chemoembolization.

This review article examines the basic principle underlying trans-arterial chemoembolization (TACE) used for treating unrespectable liver cancer with discussion on the barriers that are present for efficient drug delivery with suggestions on methods that may be used to overcome these barriers and hence enhance the efficacy of the technique. Current drugs used with TACE along with inhibitors of neovascularisation are briefly discussed. It also compares the conventional method of chemoembolization with TACE and rationalizes why there is not much of a difference between the two methods on treatment efficacy. Further it also suggests alternative methods of drug delivery that may be used instead of TACE. Additionally, it discusses the disadvantages on using non degradable microspheres with recommendations for degradable microspheres within 24 hours to overcome rebound neovascularisation owing to hypoxia. Finally, the review examines some of the biomarkers that are used to assess treatment efficacy with indication that non-invasive and sensitive biomarkers should be identified for routine screening and early detection. The review concludes that, if the current barriers present in TACE can be overcome along with the use of degradable microspheres and efficient biomarkers for monitoring efficacy, then a more robust treatment would emerge that may even serve as a cure.

Physical and chemical factors affecting the loading and release of bromelain from DC beads.

Doxorubicin loaded DC beads (microspheres) has been used for treating un-resectable tumours by transarterial chemoembolization (TACE). We have shown that bromelain, an enzyme from the pineapple plant, enhances the cytotoxic effect of a number of chemotherapeutic drugs and in an earlier study we have demonstrated that it can be loaded into DC beads. Therefore, in the current study we have investigated how certain physical and chemical parameters affect its loading and release for future development of DC beads in cancer therapy. Aliquots of 40-60 µL of DC beads (100-300 µm) were treated to bromelain in distilled water and various parameters such as pH of solution, bromelain concentration, temperature, loading period, presence/absence of agitation and the cytotoxic effect of bromelain loaded beads were investigated. Further release kinetics was also studied with additional investigation of pH effect on the proteolytic activity of bromelain. Results indicate that higher loading of bromelin was achieved in the beads at lower pH, higher concentration of bromelain, with agitation, 24 hours loading and ambient room temperature. Proteolytic activity of bromelain was maximal at pH 4.5 whilst cytotoxicity was at par if not better in the bromelain loaded DC beads. Release kinetics indicated that bromelain can be delivered over several hours. Hence, we conclude that bromelain can be loaded more efficiently with manipulation of certain parameters with noticeable cytotoxicity in tumour cells.

A novel method for potentiation of chemotherapy in soft tissue sarcomas with BromAc.

Single-agent doxorubicin currently forms part of standard care for patients with sarcomas. However, efficacy is limited by the presence of dose-dependent cardiotoxicity and toxicity to renal, hepatic, and neurological systems. Therefore, there is a pressing need for novel drug regimens which can provide increased efficacy and safety. BromAc is a novel drug combination developed as a mucolytic agent which has demonstrated anticancer activity both in vitro and in vivo in several cancers. Here, we investigated the efficacy of BromAc in combination with doxorubicin for four subtypes of sarcoma. Cell proliferation, alongside western blot for a variety of cell cycle, apoptosis, and autophagy biomarkers assays was performed following treatment of cell lines in vitro at various concentrations of BromAc and doxorubicin. The impact of drug treatment on MUC1 and MUC4 levels was assessed through immune-cytological methods. Drug agent synergy was assessed through the Chou-Talalay framework. BromAc treatment in combination with doxorubicin was more efficacious than single-agent doxorubicin, with synergistic effects observed. The immuno-cytological analysis demonstrated significant mucin depletion following treatment with BromAc and doxorubicin used in combination, providing a potential mechanistic underpinning for the observed anticancer effects.

Comparison of proteolytic, cytotoxic and anticoagulant properties of chromatographically fractionated bromelain to un-fractionated bromelain.

Bromelain consisting of a number of proteolytic enzymes possess anticancer and thrombotic properties. Hence, four chromatically separated fractions were examined for their proteolytic, anticancer and antithrombotic activity. Bromelain fractions were separated using ion-exchange column chromatography. Proteolytic properties were assessed using standard azocasein assay. Anticancer properties were first assessed using four different cell lines PANC-1, HEP 2B, HEP 3G and OVCAR-3 on cells grown in 96 well plates. Subsequently, fraction 2 and fraction 3 combined with gemcitabine were tested in ASPC-1 cells. Then cytotoxicity of fraction 3 was compared to bromelain in combination with doxorubicin and N-acetylcysteine on HEP G2 and HEP 3B cells. Finally, the anticoagulation effect of fraction 3 or bromelain combined with N-acetylcysteine was evaluated using human blood. Fraction 3 showed the highest proteolytic activity (5% greater than standard bromelain) whilst others were less active. Cytotoxicity as assessed by IC50 indicated fraction 3 to be the most potent whilst the others did not follow their proteolytic potency order. OVCAR-3 was the most sensitive amongst the cell lines. Fraction 3 showed higher potency in combination with gemcitabine in ASPC-1 cells compared to fraction 2. Similarly, fraction 3 in combination with doxorubicin showed higher toxicity when compared to bromelain. Fraction 3 or bromelain only showed thrombolytic activity in combination with N-acetylcysteine. Fraction 3 may be developed for clinical use since it showed better cytotoxicity compared to bromelain.

Addition of bromelain and acetylcysteine to gemcitabine potentiates tumor inhibition in vivo in human colon cancer cell line LS174T.

The combinations of Bromelain and Acetylcysteine (BromAc®) with cytotoxics such as Gemcitabine, 5-Fluorouracil or Oxaliplatin have shown a dramatic reduction in IC50 values in a variety of cancers, including colon cancer, suggesting the possibility of effective treatment without undesired side effects. In the current study, we investigated whether a similar effect is present in vivo using the colorectal cell line LS174T. Animals after acclimatization were randomized and allocated equally in the groups for the different studies (safety, dose-escalation, and efficacy). Drugs were delivered by the intraperitoneal route and animals were monitored for wellbeing. Separately, an efficacy study was conducted with intraperitoneal drug delivery after intraperitoneal tumor induction. At the termination of the experiment, tumors and other tissues were collected for evaluation. BromAc® was safe when delivered intraperitoneally in a rat model at the concentrations used. Subsequent investigations of these adjuvants in combination with Gemcitabine, Oxaliplatin, and 5-Fluorouracil in mice were also proven to be safe. Preliminary efficacy studies with Oxaliplatin and 5-Fluorouracil on tumor growth (LS174T) were negative. Gemcitabine was assessed with BromAc® showing an almost 71% tumor inhibition compared to controls. This in vivo study indicates that Gemcitabine at 2 mg/kg in combination with BromAc® 3 mg/300 mg/Kg was effective and safe, supporting its potential for future clinical application.

Bromelain and acetylcysteine (BromAc®) alone and in combination with gemcitabine inhibit subcutaneous deposits of pancreatic cancer after intraperitoneal injection.

Gemcitabine (GEM) is commonly chosen for treating pancreatic cancer. However, its use is limited by toxicity. Earlier in vitro studies with GEM in combination with Bromelain (Brom) and Acetylcysteine (Ac) indicated a substantial reduction in IC50. In this study, immunocytochemistry and Western blot were used to explore the mechanistic effects of Brom and Ac (BromAc®) in vitro. Then, we explored the efficacy and safety of BromAc® only and with GEM in a pancreatic cancer model in vivo. Immunocytochemistry results revealed a reduction in both MUC1 and MUC4 post-treatment. There was a decrease in VEGF, MMP-9, NF-κß and cleavage of PARP. There was also a decrease in the cell cycle regulators Cyclin B and D as well as TGF-ß and the anti-apoptotic Bcl-2. In vivo, the low and high doses of BromAc® alone and with chemotherapy agents were safe. A very significant reduction in pancreatic tumour volume, weight, and ki67 were seen with BromAc® therapy and was equal to treatment with GEM alone and better than treatment with 5-FU. In addition, tumour density was significantly reduced by BromAc®. In conclusion, the anticancer effect of BromAc® is probably related to its mucin depletion activity as well as its effect on proteins involved in cell cycle arrest, apoptosis and modulation of the tumour microenvironment. The in vivo results are encouraging and are considered the first evidence of the efficacy of BromAc® in pancreatic cancer. These results also provide some mechanistic leads of BromAc®.

The effect of intraperitoneal administration of BromAc on blood parameters: phase 1 study.

Intraperitoneal administration of BromAc (bromelain + acetylcysteine) is currently undergoing a phase 1 clinical trial for pseudomyxoma peritonei at our institution. This study reports on analysis of routine blood parameters before and after treatment for a series of 25 patients in this trial. Blood parameters assessed included full blood count, electrolytes, urea, and creatinine, liver function tests, coagulation studies, as well as inflammatory markers (CRP). Certain parameters such as CRP, and white cell count, were significantly elevated after treatment whilst serum albumin level was reduced indicating an inflammatory reaction. However, liver enzymes, coagulation studies, and other parameters were not affected. Therefore, there are no additional safety signals evident upon analysis of routine blood parameter testing.

Photothermolysis of sebaceous glands in human skin ex vivo with a 1,708 nm Raman fiber laser and contact cooling.

BACKGROUND AND OBJECTIVES: Wavelengths near ∼1,720 nm are of interest for targeting fat/lipid-rich tissues due to the high absorption coefficient of human fat and low water scattering and absorption. In this study, a 1,708 nm laser was built and shown to selectively target fat/lipid adjacent to porcine heart and dermis and then used to damage dermal sebaceous glands in human skin. STUDY DESIGN AND MATERIALS: An all-fiber 1,708 nm laser with ∼4 W maximum power was designed and built. Selectivity for targeting fat/lipid was studied by exposing porcine heart and skin tissue cross-sections to the 1,708 nm laser. Human skin treatments to damage sebaceous glands were performed both with and without cold window cooling. Histochemical evaluation on the frozen sections was performed using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. RESULTS: Histochemical analysis of porcine tissue cross-sections showed that 1,708 nm laser can selectively damage pericardial fat(heart) and subcutaneous fat(skin) with little to no damage to the myocardium and the dermis, respectively. In human skin, histochemical evaluation without contact cooling showed damage to both epidermis and dermis. With cooling, epidermis was spared and damage was observed in dermis extending ∼0.4-1.65 mm from the skin surface at an average laser fluence of ∼80 J/cm(2). Selective damage of sebaceous glands was suggested but not definitively demonstrated. CONCLUSIONS: We have developed an all-fiber 1,708 nm laser capable of damaging majority of the sebaceous glands in the dermis and thus may have potential applications in the treatment of conditions such as acne vulgaris whose pathophysiology involves disorders of sebaceous glands.

Kinetic properties of ASC protein aggregation in epithelial cells.

Apoptosis-associated speck-like protein with CARD domain (ASC), an adaptor protein composed of caspase recruitment and pyrin domains, can efficiently self-associate to form a large spherical structure, called a speck. Although ASC aggregation is generally involved with both inflammatory processes and apoptosis, the detailed dynamics of speck formation have not been characterized. In this report, speck formation in HeLa cells transfected with ASC is examined by time-lapse live-imaging by confocal laser scanning microscopy. The results show that ASC aggregation is a very rapid and tightly regulated process. Prior to speck formation, soluble ASC aggregation is a low probability event, and the affinity of ASC subunits for one another is very low. Following a speck nucleation event, the affinity for further addition of ASC subunits increases dramatically, and aggregation is a highly energetically favorable reaction (Gibbs free energy approximately -40 kJ/mol). This leads to a rapid depletion of soluble ASC, making it highly unlikely that a second speck will form inside the same cell and assuring that speck formation is "all or none," with a well-defined end point. Comparison with kinetic models of the aggregation process indicates diffusion, instead of active transport, is the dominant process for speck growth. Though speck formation and aggresome formation share some properties, we show that the two processes are distinct.

Mid-infrared absorption spectroscopy and differential damage in vitro between lipids and proteins by an all-fiber-integrated supercontinuum laser.

We identify and differentially damage lipids and proteins using wavelengths between 2.6 and 3.8 mum from a fiber-based supercontinuum (SC) laser. Absorption spectroscopy of the constituents of normal artery and atherosclerotic plaque, including adipose tissue, macrophages and foam cells, are measured by a SC laser in the mid-infrared. By using the laser light within the C-H fatty acid and cholesterol esters absorption band, we also demonstrate differential damage of lipid-rich adipose tissue without damaging the protein-rich blood vessel wall. The experiments use a novel SC laser that is all-fiber-integrated with no moving parts, covers a continuous spectrum ranging from approximately 0.8 to beyond 4.2 microm, and outputs a time-averaged power scalable up to 10.5 W.