Piyali Dasgupta

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology.

Neovascular diseases of the retina, such as diabetic retinopathy (DR) and age -related macular degeneration (AMD), are proliferative retinopathies involving the growth of new blood vessels on the retina, which in turn causes impairment and potential loss of vision. A drawback of conventional angiogenesis assays is that they are not representative of the angiogenic processes in the retina. In the retina, the new blood vessels grow (from pre-existing blood vessels) and migrate into a non-perfused region of the eye including the inner limiting membrane of the retina and the vitreous, both of which contribute to vision loss. The Matrigel Duplex Assay (MDA) measures the migration of angiogenic capillaries from a primary Matrigel layer to a secondary Matrigel layer, which resembles the pathological angiogenesis in AMD and DR. The methodology of MDA is comprised of two steps. In the first step, the human retinal microvascular endothelial cells (HRMECs) are mixed with phenol red-containing Matrigel (in a 1:1 ratio) and seeded in the center of an 8 -well chamber slide. After 24 h, a second layer of phenol red-free Matrigel is overlaid over the first layer. Over the course of the next 24 h, the HRMECs invade from the primary Matrigel layer to the secondary layer. Subsequently, the angiogenic sprouts are visualized by brightfield phase contrast microscopy and quantified by ImageJ software. The present manuscript measures the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in primary HRMECs using the MDA. The MDA may be used for multiple applications like screening anti-angiogenic drugs, measuring the pro-angiogenic activity of growth factors, and elucidating signaling pathways underlying retinal angiogenesis in normal and disease states.

The invasion of tumor cells into the neighboring blood vessels and lymph nodes is a vital step for distant metastasis. Traditionally, the invasive activity of growth factors (or the anti-invasive activity of drugs) is measured with the Boyden chamber assay. However, this assay has a few disadvantages like poor physiological relevance of transwell inserts and an inability to control chemokine gradients. The Boyden chamber assay is one of the most prevalent methods to measure the invasion of cancer cells. It would be advantageous to develop another assay that could validate the results of the Boyden chamber assay. With this in mind, our laboratory developed the spherical invasion assay (SIA) to measure the pro-invasive activity of human cancer cells. The SIA also circumvents some of the drawbacks of the Boyden chamber assay. The present manuscript measures the anti-invasive activity of the Src kinase inhibitor PP2 in A549 human non-small cell lung carcinoma (NSCLC) cells using the SIA. The SIA protocol is comprised of two steps. In the first step, A549 human NSCLC cells (treated or not with PP2) were mixed with Matrigel and seeded in the middle of an eight-well chamber slide. After 24 h, a second layer of Matrigel was overlaid over the first layer. Over the course of the next 24 h, the A549 cells invade from the primary to the secondary Matrigel layers. Subsequently, the cells are visualized by phase-contrast microscopy and the images obtained are quantified using ImageJ to calculate the anti-invasive activity of PP2 in A549 cells. The results of the SIA correlate well with Boyden chamber assays. The SIA may be adapted for multiple experimental designs, such as drug screening (to combat invasion and metastasis), measuring the pro-invasive activity of growth factors, and elucidating the signaling pathways underlying the pro-invasive/anti-invasive activity of biological modifiers.Graphic Diagrammatic illustration of the spherical invasion assay (Hurley et al., 2017). A. The first layer is comprised of human cancer cells mixed in a 1:1 suspension with Phenol Red containing Matrigel (represented as LAYER 1 in the figure). After 24 h, the cancer cells grow and extend up to the boundary of this first layer. B. A second layer of 1:1 solution Phenol Red-free Matrigel, in Phenol Red-free RPMI (represented as LAYER 2 in the figure) is added on top of the first Matrigel spot. The cells are incubated for 24 h at 37°C. C. Over these 24 h, the cancer cells invade from the primary layer into the secondary Matrigel layer. The chamber slides are observed by phase-contrast microscopy. D. A representative photograph of the images obtained by the SIA is shown. The black arrow indicates the cancer cells invading into the second layer of Matrigel. The dotted line represents the interface between the two layers. The distance to which the cells have traveled (into the secondary Matrigel layer) is measured at ten sites (for each photograph) in a randomized double-blind fashion by three independent observers, using NIH ImageJ Version 1.47. This process is repeated for three separate photographic fields per sample., [摘要] 肿瘤细胞侵入邻近血管和淋巴结是远处转移的关键步骤。传统上，生长因子的侵袭活性（或药物的抗侵袭活性）是用 Boyden 室测定法测量的。然而，这种测定有一些缺点，如transwell插入物的生理相关性差和无法控制趋化因子梯度。 Boyden 室试验是测量癌细胞侵袭的最普遍的方法之一。开发另一种可以验证 Boyden 室试验结果的试验将是有利的。考虑到这一点，我们的实验室开发了球形侵袭试验 (SIA) 来测量人类癌细胞的促侵袭活性。 SIA 还规避了 Boyden 室检测的一些缺点。本手稿使用 SIA 测量了Src激酶抑制剂 PP2 在 A549 人非小细胞肺癌 (NSCLC) 细胞中的抗侵袭活性。 SIA 协议由两个步骤组成。在第一步中，将 A549 人 NSCLC 细胞（用 PP2 处理或未处理）与 Matrigel 混合并接种在八孔室载玻片的中间。 24 小时后，将第二层 Matrigel覆盖在第一层上。在接下来的 24 小时内，A549 细胞从初级基质胶层侵入到次级基质胶层。随后，通过相差显微镜观察细胞，并使用 ImageJ 对获得的图像进行量化，以计算 PP2 在 A549 细胞中的抗侵袭活性。 SIA 的结果与 Boyden 室检测的相关性很好。 SIA 可适用于多种实验设计，例如药物 筛选（以对抗侵袭和转移），测量生长因子的促侵袭活性，并阐明生物修饰剂的促侵袭/抗侵袭活性背后的信号通路。图文摘要：球形侵袭试验的图解说明（ Hurley等人，2017 年） 。一个。第一层由人类癌细胞组成，以 1:1 的比例混合在含有酚红的基质胶（图中表示为 LAYER 1）中。 24 小时后，癌细胞生长并延伸到第一层的边界。 B. 在第一个 Matrigel 点的顶部添加第二层 1:1 溶液 Phenol Red-free Matrigel，在 Phenol Red-free RPMI 中（在图中表示为 LAYER 2）。将细胞在 37 °C下孵育 24 小时。 C. 在这 24 小时内，癌细胞从初级层侵入到次级基质胶层。通过相差显微镜观察室载玻片。 D. 显示了 SIA 获得的图像的代表性照片。黑色箭头表示癌细胞侵入 Matrigel 第二层。虚线表示两层之间的界面。使用 NIH ImageJ 版本 1.47，由三位独立观察者以随机双盲方式在十个位置（对于每张照片）测量细胞行进的距离（进入次级基质胶层）。对每个样本的三个单独的摄影场重复该过程。[背景] 肿瘤侵袭是指癌细胞穿透基底膜进入周围基质、邻近血管和淋巴结的过程（ Stuelten 等人，2018 年；巴格班 等人，2020 年）。肿瘤侵袭是人类癌症转移的必要步骤（巴格班 等人，2020 年）。在转移的众多步骤中，许多治疗药物都针对肿瘤侵袭过程来抑制肿瘤的远处扩散（ Gandalovicova）。...

Ovarian cancer is a fatal gynecological cancer because of a lack of early diagnosis, which often relapses as chemoresistant. Trichodermin, a trichothecene first isolated from Trichoderma viride, is an inhibitor of eukaryotic protein synthesis. However, whether trichodermin is able to suppress ovarian cancer or not was unclear. In this study, trichodermin (0.5 µM or greater) significantly decreased the proliferation of two ovarian cancer cell lines A2780/CP70 and OVCAR-3. Normal ovarian IOSE 346 cells were much less susceptible to trichodermin than the cancer cell lines. Trichodermin predominantly inhibited ovarian cancer cells by inducing G0/G1 cell cycle arrest rather than apoptosis. Trichodermin decreased the expression of cyclin D1, CDK4, CDK2, retinoblastoma protein, Cdc25A, and c-Myc but showed little effect on the expression of p21^Waf1/Cip1, p27^Kip1, or p16^Ink4a. c-Myc was a key target of trichodermin. Trichodermin regulated the expression of Cdc25A and its downstream proteins via c-Myc. Overexpression of c-Myc attenuated trichodermin’s anti-ovarian cancer activity. In addition, trichodermin decelerated tumor growth in BALB/c nude mice, proving its effectiveness in vivo. These findings suggested that trichodermin has the potential to contribute to the treatment of ovarian cancer.

Capsaicin displays robust growth-inhibitory activity in multiple human cancers. However, the feasibility of capsaicin as a clinically relevant anticancer drug is hampered by its adverse side effects. This concern has led to extensive research focused on the isolation and synthesis of second-generation nonpungent capsaicin analogues with potent antineoplastic activity. A major class of nonpungent capsaicin-like compounds belongs to the N-acyl-vanillylamide (N-AVAM) derivatives of capsaicin (hereafter referred as N-AVAM capsaicin analogues). This perspective discusses the isolation of N-AVAM capsaicin analogues from natural sources as well as their synthesis by chemical and enzymatic methods. The perspective describes the pharmacokinetic properties and anticancer activity of N-AVAM capsaicin analogues. The signaling pathways underlying the growth-inhibitory effects of N-AVAM capsaicin analogues have also been highlighted. It is hoped that the insights obtained in this perspective will facilitate the synthesis of a second generation of N-AVAM capsaicin analogues with improved stability and growth-suppressive activity in human cancer.

Capsaicin (trans-8-methyl-N-vanillyl-6-noneamide) is a hydrophobic, lipophilic vanilloid phytochemical abundantly found in chili peppers and pepper extracts. Several convergent studies show that capsaicin displays robust cancer activity, suppressing the growth, angiogenesis and metastasis of several human cancers. Despite its potent cancer-suppressing activity, the clinical applications of capsaicin as a viable anti-cancer drug have remained problematic due to its poor bioavailability and aqueous solubility properties. In addition, the administration of capsaicin is associated with adverse side effects like gastrointestinal cramps, stomach pain, nausea and diarrhea and vomiting. All these hurdles may be circumvented by encapsulation of capsaicin in sustained release drug delivery systems. Most of the capsaicinbased the sustained release drugs have been tested for their pain-relieving activity. Only a few of these formulations have been investigated as anti-cancer agents. The present review describes the physicochemical properties, bioavailability, and anti-cancer activity of capsaicinsustained release agents. The asset of such continuous release capsaicin formulations is that they display better solubility, stability, bioavailability, and growth-suppressive activity than the free drug. The encapsulation of capsaicin in sustained release carriers minimizes the adverse side effects of capsaicin. In summary, these capsaicin-based sustained release drug delivery systems have the potential to function as novel chemotherapies, unique diagnostic imaging probes and innovative chemosensitization agents in human cancers.