Understanding cancer stem cells and plasticity: Towards better therapeutics.

The ability of cancer cells to finally overcome various lines of treatment in due course has always baffled the scientific community. Even with the most promising therapies, relapse is ultimately seen, and this resilience has proved to be a major hurdle in the management of cancer. Accumulating evidence now attributes this resilience to plasticity. Plasticity is the ability of cells to change their properties and is substantial as it helps in normal tissue regeneration or post-injury repair processes. It also helps in the overall maintenance of homeostasis. Unfortunately, this critical ability of cells, when activated incorrectly, can lead to numerous diseases, including cancer. Therefore, in this review, we focus on the plasticity aspect with an emphasis on cancer stem cells (CSCs). We discuss the various forms of plasticity that provide survival advantages to CSCs. Moreover, we explore various factors that affect plasticity. Furthermore, we provide the therapeutic implications of plasticity. Finally, we provide an insight into the future targeted therapies involving plasticity for better clinical outcomes.

Survivin Inhibition by Piperine Sensitizes Glioblastoma Cancer Stem Cells and Leads to Better Drug Response.

Glioblastoma multiforme (GBM) cancer stem cells (GSCs) are one of the strongest contributing factors to treatment resistance in GBM. Identification of biomarkers capable of directly affecting these cells within the bulk tumor is a major challenge associated with the development of new targeting strategies. In this study, we focus on understanding the potential of the multifunctional extraordinaire survivin as a biomarker for GSCs. We analyzed the expression profiles of this gene using various publicly available datasets to understand its importance in stemness and other cancer processes. The findings from these studies were further validated using human GSCs isolated from a GBM cell line. In these GSCs, survivin was inhibited using the dietary phytochemical piperine (PIP) and the subsequent effects on stemness, cancer processes and Temozolomide were investigated. In silico analysis identified survivin to be one of the most significant differentially regulated gene in GSCs, in comparison to common stemness markers. Further validation studies on the isolated GSCs showed the importance of survivin in stemness, cancer progression and therapy resistance. Taken together, our study identifies survivin as a more consistent GSC marker and also suggests the possibility of using survivin inhibitors along with standard of care drugs for better therapeutic outcomes.

Photobiomodulation invigorating collagen deposition, proliferating cell nuclear antigen and Ki67 expression during dermal wound repair in mice.

The present investigation focuses on understanding the role of photobiomodulation in enhancing tissue proliferation. Circular excision wounds of diameter 1.5 cm were created on Swiss albino mice and treated immediately with 2 J/cm2 and 10 J/cm2 single exposures of the Helium-Neon laser along with sham-irradiated controls. During different days of healing progression (day 5, day 10, and day 15), the tissue samples upon euthanization of the animals were taken for assessing collagen deposition by Picrosirius red staining and cell proliferation (day 10) by proliferating cell nuclear antigen (PCNA) and Ki67. The positive influence of red light on collagen synthesis was found to be statistically significant on day 10 (P < 0.01) and day 15 (P < 0.05) post-wounding when compared to sham irradiation, as evident from the image analysis of collagen birefringence. Furthermore, a significant rise in PCNA (P < 0.01) and Ki67 (P < 0.05) expression was also recorded in animals exposed to 2 J/cm2 when compared to sham irradiation and (P < 0.01) compared to the 10 J/cm2 treated group as evidenced by the microscopy study. The findings of the current investigation have distinctly exhibited the assenting influence of red laser light on excisional wound healing in Swiss albino mice by augmenting cell proliferation and collagen deposition.

Pseudomonas aeruginosa virulence proteins pseudolysin and protease IV impede cutaneous wound healing.

The intricate biological process of cutaneous wound healing is achieved through precise and highly programmed events. Dermal fibroblasts and keratinocytes play a significant role in the process of reepithelialization during wound healing. Pathogenic bacteria such as Pseudomonas aeruginosa (P. aeruginosa) may delay the proliferative phase of wound repair by secreting their proteins leading to delayed or impaired wound healing. We have analyzed three virulent strains of P. aeruginosa isolated from the wound environment which also differed in their ability to produce biofilms. Mass spectrometric analysis of differentially expressed secreted proteins by three virulent strains of P. aeruginosa revealed peptides from pseudolysin and protease IV expressed from lasB and prpL genes. Pseudolysin and protease IV recombinant proteins were tested for their ability to modulate wound healing in several cell types of wound microenvironment in in vitro and in vivo models. Both pseudolysin and protease IV inhibited migration and survival of fibroblasts, keratinocytes, and endothelial cells. In three dimensional spheroid endothelial models and matrigel assays these proteins impeded sprouting and tube formation. In a mouse model of excision wound, pseudolysin and protease IV treatment showed reduced collagen content, inhibited neovascularization and epithelialization, and delayed wound contraction. Furthermore, pseudolysin and protease IV treatment resulted in a significant increase in plasma IL-6 levels when compared to vehicle control and control, suggesting the induction of a state of prolonged inflammation. Taken together, our data indicate pseudolysin and protease IV secreted from biofilm producing and antibiotic resistant P. aeruginosa in wound microenvironment produce both local and systemic effects that is detrimental to the maintenance of tissue homeostasis. Hence, these proteins may serve as potential therapeutic targets toward better clinical management of wounds.

Thread integrated smart-phone imaging facilitates early turning point colorimetric assay for microbes.

This study employs a commercial multifilament cotton thread as a low-cost microbial identification assay integrated with smartphone-based imaging for high throughput and rapid detection of pathogens. The thread device with inter-twined fibers was drop-cast with test media and a pH indicator. The target pathogens scavenge the media components with different sugars and release acidic by-products, which in turn act as markers for pH-based color change. The developed thread-based proof-of-concept was demonstrated for the visual color detection (red to yellow) of Candida albicans (≈16 hours) and Escherichia coli (≈5 hours). Besides that, using a smart-phone to capture images of the thread-based colorimetric assay facilitates early detection of turning point of the pH-based color change and further reduces the detection time of pathogens viz. Candida albicans (≈10 hours) and Escherichia coli (≈1.5 hours). The reported thread and smartphone integrated image analysis works towards identifying the turning point of the colorimetric change rather than the end-point analysis. Using this approach, the interpretation time can be significantly reduced compared to the existing conventional microbial methods (≈24 hours). The thread-based colorimetric microbial assay represents a ready-to-use, low-cost and straightforward technology with applicability in resource-constrained environments, surpassing the need for frequent fresh media preparation, expensive instrumentation, complex fabrication techniques and expert intervention. The proposed method possesses high scalability and reproducibility, which can be further extended to bio(chemical) assays.

Probing endogenous collagen by laser-induced autofluorescence in burn wound biopsies: A pilot study.

The focus of the current study was to interrogate the predictive potential of laser-induced autofluorescence (LIAF) by objectively assessing collagen synthesis in burn wound granulation tissues ex vivo. Prior grafting, granulation tissues (20 samples) following burn injury were collected from 17 subjects of age range 18 to 60 years with patient/donor consent and the corresponding autofluorescence spectra were recorded at 325 nm He-Cd laser (≈2 mW) excitations. The resulting endogenous collagen intensity from the above tissue samples was computed by normalizing the nicotinamide adenine dinucleotide levels. In addition, the hydroxyproline content was also estimated biochemically from the same granulation tissues. A comparative assessment of both LIAF and biochemical estimations for endogenous collagen by hydroxyproline resulted in strong positive correlation among them. The above relevant observations suggest that LIAF is equally informative as that of biochemical estimations, in evaluating endogenous collagen content in wound granulation tissues. Thus, it can be concluded that LIAF has the predictive potential, as a noninvasive objective tool to measure the endogenous collagen levels in wound biopsy tissues and provide complementary data conducive for making clinical decisions.

Laser-induced autofluorescence-based objective evaluation of burn tissue repair in mice.

Management of burn injuries are a growing concern, especially in determining the progression of healing. Several techniques are being practiced in clinics and have been considered all-time standard approaches to determine pre- and post-treatment outcomes of a healthy healing. However, these kinds of methods involve repeated biopsies and thereby hindering tissue repair. In view of this, our perspective was to develop a non-invasive tool in an attempt to provide a solution to determine the progression of healing, in vivo. Hence, the present study was designed to investigate the ability of laser-induced fluorescence (LIF) to monitor the variations in collagen intensity at various time points (0, 2, 6, 12, 18, 24, and 30 days) during burn tissue repair in mice, post low-power laser therapy (LPLT). The spectral findings demonstrated a significant change in collagen intensity as observed on day 24 (p < 0.05) and 30 (p < 0.01), when treated with LPLT (830 nm 3 J/cm2) as compared to untreated control. From the observation, it was evident that the LIF could objectively monitor the progression of burn tissue repair in vivo.

In vitro culture responses, callus growth and organogenetic potential of brinjal (Solanum melongena L.) to He-Ne laser irradiation.

The present investigation was designed to analyze the influence of Helium-Neon (He-Ne 632.8nm) laser irradiation on defense enzymes, proline content and in vitro responses of callus induction, shoot initiation and on plantlet regeneration potential of brinjal. The seeds of Mattu Gulla (Solanum melongena L.) were irradiated with 20, 25 and 30J/cm2 of He-Ne laser followed by surface sterilization and sprouted on Murashige and Skoog medium without plant growth regulators. The activity of defense enzymes, proline content and the organogenetic potential of hypocotyl, leaf and shoot tip explants were determined from thirty day old seedlings. During seed germination, most of the seedlings showed normal two cotyledons whereas small number of seedlings showed tricotyledonous at 20J/cm2 treatment and no other morphological abnormalities were observed during further growth and development. There was no substantial variation was noted in both ß-1,3-glucanase and chitinase activity as well as proline content which proves the He-Ne laser irradiation does not causes any stresses for the plant. The in vitro culture of hypocotyl, leaf and shoot tip explants from laser irradiated seedlings showed differential responses as compared to un-irradiated control. The laser induced enhancement of callus induction, growth rate of callus tissues and shoot tip, percentage of responses of shoot and root initiation, days to shoot and root initiation, shoots formed per callus, number of roots per shoots, length of roots and nuclear DNA content of in vitro raised plants were evaluated. Among the tested laser doses (20, 25 and 30J/cm2), 25J/cm2 showed significant biostimulatory effect over un-irradiated control seedlings. The present observations reveal and endorsed our earlier reports with substantial enhancement of in vitro and ex vitro by He-Ne laser irradiation.

Photo-biomodulatory response of low-power laser irradiation on burn tissue repair in mice.

The present work reports the photo-biomodulatory effect of red (632.8 nm) and near infrared (785 and 830 nm) lasers on burn injury in Swiss albino mice. Animals were induced with a 15-mm full thickness burn injury and irradiated with various fluences (1, 2, 3, 4, and 6 J/cm2) of each laser wavelength under study having a constant fluence rate (8.49 mW/cm2). The size of the injury following treatment was monitored by capturing the wound images at regular time intervals until complete healing. Morphometric assessment indicated that the group treated with 3-J/cm2 fluence of 830 nm had a profound effect on healing as compared to untreated controls and various fluences of other wavelengths under study. Histopathological assessment of wound repair on treatment with an optimum fluence (3 J/cm2) of 830 nm performed on days 2, 6, 12, and 18 post-wounding resulted in enhanced wound repair with migration of fibroblasts, deposition of collagen, and neovascularization as compared to untreated controls. The findings of the present study have clearly demonstrated that a single exposure of 3-J/cm2 fluence at 830-nm enhanced burn wound healing progression in mice, which is equivalent to 5 % povidone iodine treatment (reference standard), applied on a daily basis till complete healing.

Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence.

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.

Influence of helium-neon laser irradiation on seed germination in vitro and physico-biochemical characters in seedlings of brinjal (Solanum melongena L.) var. Mattu Gulla.

In the present study, the seeds of brinjal (Solanum melongena L.) var. Mattu Gulla were irradiated with single exposure of He-Ne laser at different doses of 5-40 J cm(-2) and germinated aseptically. Thirty day old seedlings were harvested and the germination, growth, physiological and biochemical parameters were estimated and compared with un-irradiated control seedlings. A significant enhancement in growth characters were noted with respect to length, fresh and dry weight of shoots and roots. In addition, chlorophyll (a and b), carotenoid content, anthocyanin and amylases (α and ß) activities were found to be altered. Significant alterations in percentage of seed germination (P < 0.001) and time to 50% germination (P < 0.001) were observed in the irradiated seeds compared with the un-irradiated controls. In conclusion, the results of the present study demonstrated that low dose (5-30 J cm(-2) ) of He-Ne laser irradiation enhanced the germination process and altered growth, by positively influencing physiological and biochemical parameters of the brinjal seedlings compared with un-irradiated control under in vitro conditions.

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT).

The present study focuses on the evaluation of the effect of He-Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post-wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non-illuminated controls.

Effect of laser dose and treatment schedule on excision wound healing in diabetic mice.

The present study was undertaken to evaluate a He-Ne laser (632.8 nm; 7 mW; 4.02 mW cm(-2); 15 mm spot size) dose and the treatment schedule on diabetic wound healing in a mouse model. Circular wounds of 15 mm diameter were created on streptozotocin induced diabetic Swiss albino mice, and were uniformly illuminated with the single exposure of various He-Ne laser doses of 1, 2, 3, 4 and 5 J cm(-2) respectively. Further, the treatment schedule was also optimized by exposing the wounds with 3 J cm(-2) at 0, 24 h, 48 h postwounding. Contraction kinetics, mean area under the curve and the mean healing time of the wounds were computed along with the collagen and the glucosamine levels in the wound ground tissues at various postwounding treatment schedules. Results of this study indicated that the single exposure of 3 J cm(-2) laser dose applied immediately after the wounding caused a significant reduction in the mean area under the curve and the mean healing time along with the elevated levels of collagen and glucosamine contents in the tissue compared to the controls. In conclusion, He-Ne laser dose of 3 J cm(-2) applied immediately after the wounding has demonstrated optimum wound healing compared to the other doses and treatment schedules.

Evaluation of pharmacokinetic, biodistribution, pharmacodynamic, and toxicity profile of free juglone and its sterically stabilized liposomes.

The present study was aimed to formulate and compare the pharmacokinetic, biodistribution, pharmacodynamic, and toxicity profiles of free 5-hydroxy-1,4-naphthoquinone (juglone) with sterically stabilized liposomal form. The liposomes were optimized for size, zeta potential, entrapment efficiency (EE), and in vitro release properties. The optimized formulation had a mean size, zeta potential, and EE value of 137.1 nm, -43.1 mV, and 67.2%, respectively. In vitro release studies showed biphasic pattern with initial burst followed by sustained release over the study period, releasing about 61% after 24 h. In vitro cytotoxicity studies against melanoma cells indicated that liposomal juglone was more toxic than free juglone. Free juglone had short plasma half-life of about 2 h, whereas liposomal juglone exhibited significantly improved pharmacokinetics with a 12-fold increase in plasma half-life. Further, biodistribution studies indicated rapid renal elimination of free juglone, evidenced by its significant localization in kidneys. Conversely, the accumulation of liposomal juglone in kidneys reduced significantly with enhanced tumor localization, thereby resulting in enhanced antitumor activity. The histological studies revealed lower levels of nephrotoxicity for liposomal juglone compared with that of free juglone. To conclude, sterically stabilized liposomes could be a promising approach for the intravenous delivery of hydrophobic compounds such as juglone.

Autofluorescence of osteoporotic mouse femur bones: a pilot study.

OBJECTIVE: The objective of this study was to evaluate the efficacy of laser-induced fluorescence (LIF) in combination with principal component analysis (PCA) to characterize osteoporotic conditions induced by ovariectomy (OVX) in mice. BACKGROUND DATA: The dual energy X-ray absorptiometry is the gold standard methodology routinely used to diagnose osteoporosis. In recent years, the use of LIF to characterize human disease and to aid in diagnosis has shown great promise. However, this technique has not been much exploited for monitoring osteoporosis. MATERIALS AND METHODS: Swiss albino mice were sacrificed 2, 3, 4, and 5 wk after OVX and their femur bones were excised. The same protocol was used for age-matched female controls (no OVX). The LIF spectra from different regions of the bones were recorded and compared using PCA. RESULTS: A significant change in the fluorescence pattern of osteoporotic bones compared with the control was indicated by PCA match/no-match analysis. A region-wise PCA match/no-match analysis of the spectral changes against respective region calibration sets indicated more no-matches in the fifth week bones compared with the others. Further, the spectral differences were more prominent in the proximal and distal parts of the bones. In addition to the PCA, Gaussian curve fitting was also performed on control, third week, and fifth week bone spectra to identify different spectral components in them. CONCLUSION: This preliminary study using fluorescence spectroscopy in combination with PCA clearly demonstrated osteoporotic changes in mouse femur bones at different time points after OVX, suggesting possible human applications.

Development and evaluation of fiber optic probe-based helium-neon low-level laser therapy system for tissue regeneration--an in vivo experimental study.

We report the design and development of an optical fiber probe-based Helium-Neon (He-Ne) low-level laser therapy system for tissue regeneration. Full thickness excision wounds on Swiss albino mice of diameter 15 mm were exposed to various laser doses of 1, 2, 3, 4, 6, 8 and 10 J cm(-2) of the system with appropriate controls, and 2 J cm(-2) showing optimum healing was selected. The treatment schedule for applying the selected laser dose was also standardized by irradiating the wounds at different postwounding times (0, 24 and 48 h). The tissue regeneration potential was evaluated by monitoring the progression of wound contraction and mean wound healing time along with the hydroxyproline and glucosamine estimation on wound ground tissues. The wounds exposed to 2 J cm(-2) immediately after wounding showed considerable contraction on days 5, 9, 12, 14, 16 and 19 of postirradiation compared with the controls and other treatment schedules, showing significant (P < 0.001) decrease in the healing time. A significant increase in hydroxyproline and glucosamine levels was observed for the 2 J cm(-2) irradiation group compared with the controls and other treatment groups. In conclusion, the wounds treated with 2 J cm(-2) immediately after the wounding show better healing compared with the controls.

Evaluation of high-performance liquid chromatography laser-induced fluorescence for serum protein profiling for early diagnosis of oral cancer.

The present work deals with the evaluation of a high-performance liquid chromatography laser-induced fluorescence (HPLC-LIF) technique developed in our laboratory for early detection of oral cancer from protein profiles of body fluids. The results show that protein profiles of serum samples from a given class of samples, say, normal, premalignant, or malignant, are statistically very close to each other, while profiles of members of any class are significantly different from other classes. The performance of the technique is evaluated by the use of sensitivity and specificity pairs, receiver operating characteristic (ROC) analysis, and Youden's Index. The technique uses protein profile differences in serum samples, registered by the HPLC-LIF technique. The study is carried out using serum samples from volunteers diagnosed as normal or premalignant clinically, and as malignant by histopathology. The specificities and sensitivities of the HPLC-LIF method at an ideal threshold (M-distance = 2) for normal, malignant, and premalignant classes are 100, 69.5, and 61.5%, and 86.5, 87.5, and 87.5% respectively.