Machine learning in the evaluation and prediction models of biochar application: A review.

This article reviews recent studies applying machine learning (ML) approaches to biochar applications. We first briefly introduce the general biochar production process. Various aspects are contained, including the biochar application in the elimination of heavy metals and/or organic compounds and the biochar application in environmental and economic scopes, for instance, food security, energy, and carbon emission. The utilization of ML methods, including ANN, RF, and NN, plays a vital role in evaluating and predicting the efficiency of biochar absorption. It has been proved that ML methods can validly predict the adsorption effectiveness of biochar for water heavy metals with higher accuracy. Moreover, the literature proposed a comprehensive data-driven model to forecast biochar yield and compositions under various biomass input feedstock and different pyrolysis criteria. They said a 12.7% improvement in prediction accuracy compared to the existing literature. However, it might need further optimization in this direction. In summary, this review concludes increasing studies that a well-trained ML method can sufficiently reduce the number of experiment trials and working times associated with higher prediction accuracy. Moreover, further studies on ML applications are needed to optimize the trade-off between biochar yield and its composition.

C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic Function to Mutant p53 Cancer Cells In Vitro and In Vivo.

We previously demonstrated that a synthetic monomer peptide derived from the C-terminus of p53 (aa 361−382) induced preferential apoptosis in mutant p53 malignant cells, but not normal cells. The major problem with the peptide was its short half-life (half-life < 10 min.) due to a random coil topology found in 3D proton NMR spectroscopy studies. To induce secondary/tertiary structures to produce more stability, we developed a peptide modelled after the tetrameric structure of p53 essential for activation of target genes. Starting with the above monomer peptide (aa 361−382), we added the nuclear localization sequence of p53 (aa 353−360) and the end of the C-terminal sequence (aa 383−393), resulting in a monomer spanning aa 353−393. Four monomers were linked by glycine to maximize flexibility and in a palindromic order that mimics p53 tetramer formation with four orthogonal alpha helices, which is required for p53 transactivation of target genes. This is now known as the 4 repeat-palindromic-p53 peptide or (4R-Pal-p53p). We explored two methods for testing the activity of the palindromic tetrapeptide: (1) exogenous peptide with a truncated antennapedia carrier (Ant) and (2) a doxycycline (Dox) inducer for endogenous expression. The exogenous peptide, 4R-Pal-p53p-Ant, contained a His tag at the N-terminal and a truncated 17aa Ant at the C-terminal. Exposure of human breast cancer MB-468 cells and human skin squamous cell cancer cells (both with mutant p53, 273 Arg->His) with purified peptide at 7 µM and 15 µM produced 52% and 75%, cell death, respectively. Comparatively, the monomeric p53 C-terminal peptide-Ant (aa 361−382, termed p53p-Ant), at 15 µM and 30 µM induced 15% and 24% cell death, respectively. Compared to the p53p-Ant, the exogenous 4R-pal-p53p-Ant was over five-fold more potent for inducing apoptosis at an equimolar concentration (15 µM). Endogenous 4R-Pal-p53p expression (without Ant), induced by Dox, resulted in 43% cell death in an engineered MB468 breast cancer stable cell line, while endogenous p53 C-terminal monomeric peptide expression produced no cell death due to rapid peptide degradation. The mechanism of apoptosis from 4R-Pal-p53p involved the extrinsic and intrinsic pathways (FAS, caspase-8, Bax, PUMA) for apoptosis, as well as increasing reactive oxygen species (ROS). All three death pathways were induced from transcriptional/translational activation of pro-apoptotic genes. Additionally, mRNA of p53 target genes (Bax and Fas) increased 14-fold and 18-fold, respectively, implying that the 4R-Pal-p53p restored full apoptotic potential to mutant p53. Monomeric p53p only increased Fas expression without a transcriptional or translational increase in Fas, and other genes and human marrow stem cell studies revealed no toxicity to normal stem cells for granulocytes, erythrocytes, monocytes, and macrophages (CFU-GEMM). Additionally, the peptide specifically targeted pre-malignant and malignant cells with mutant p53 and was not toxic to normal cells with basal levels of WT p53.

Gene Therapy with p14/tBID Induces Selective and Synergistic Apoptosis in Mutant Ras and Mutant p53 Cancer Cells In Vitro and In Vivo.

Any gene therapy for cancer will be predicated upon its selectivity against cancer cells and non-toxicity to normal cells. Therefore, safeguards are needed to prevent its activation in normal cells. We designed a minimal p14ARF promoter with upstream Ap1 and E2F enhancer elements and a downstream MDR1 inhibitory element, TATA box, and a transcription initiation site (hereafter p14ARFmin). The modified p14ARFmin promoter was linked to bicistronic P14 and truncated BID (tBID) genes, which led to synergistic apoptosis via the intrinsic and extrinsic pathways of apoptosis when expressed. The promoter was designed to be preferentially activated by mutant Ras and completely inhibited by wild-type p53 so that only cells with both mutant Ras and mutant p53 would activate the construct. In comparison to most p53 gene therapies, this construct has selective advantages: (1) p53-based gene therapies with a constitutive CMV promoter cannot differentiate between normal cells and cancer cells, and can be toxic to normal cells; (2) our construct does not induce p21WAF/CIPI in contrast to other p53-based gene therapies, which can induce cell cycle arrest leading to increased chemotherapy resistance; (3) the modified construct (p14ARFmin-p14-tBID) demonstrates bidirectional control of its promoter, which is completely repressed by wild-type p53 and activated only in cells with both RAS and P53 mutations; and (4) a novel combination of genes (p14 and tBID) can synergistically induce potent intrinsic and extrinsic apoptosis in cancer cells.

Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide.

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382, p53p), linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction, induced rapid apoptosis in human premalignant and malignant cell lines. Here, we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas, which express sufficient quantities of endogenous mutant or WT p53, may be restored or activated, respectively, by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system, which circumvents the blood-brain barrier.

Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143.

The p53 mutant 143Ala is a human temperature-sensitive mutant with two conformational states. To definitively determine whether the Fas signal transduction pathway and the function of the pathway are dependent on p53 status, we have established stable transfectants of p53 mutant 143Ala in two human cancer cell lines: H1299 (lung cancer line) and PC-3 (prostate cancer line), the native state of which contains null p53 status and can grow at 37 degrees C and 32.5 degrees C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis showed inhibition of the growth of cells overexpressing p53 mutant 143Ala in the wild-type p53 form at 32.5 degrees C because of induction of G0/G1 arrest. Transfected cells had increased protein expression of p21, Fas, and MDM2 at the wild-type p53 conformation at 32.5 degrees C, but not in the mutant p53 form at 37 degrees C. However, there was no change in protein expression of FADD, FAP-1, Bcl-2, or Bax at 32.5 or 37 degrees C. Assays for apoptosis demonstrated that anti-Fas antibody CH-11 and FasL induced apoptosis only in cells that overexpress p53 mutant 143Ala at 32.5 degrees C with the wild-type p53 form. Both caspase-3 and caspase-8 activities were increased by anti-Fas antibody CH-11 only in cells at 32.5 degrees C with wild-type p53. Our results demonstrated that Fas-mediated apoptosis in H1299 and PC-3 cells expressing p53 mutant 143Ala occurred only with the wild-type p53 phenotype. These results support the hypothesis that Fas-mediated apoptosis is dependent, at least partially, on the presence of a functional wild-type p53 state. This model may be a useful tool for dissecting the specific interactions between wild-type p53 and the Fas signal transduction pathway in human cancer cells.

Selective induction of apoptosis in mutant p53 premalignant and malignant cancer cells by PRIMA-1 through the c-Jun-NH2-kinase pathway.

PRIMA-1 (p53 reactivation and induction of massive apoptosis) is a chemical compound that was originally identified as a selective mutant p53-dependent growth suppressor by screening a library of low-molecular-weight compounds. However, its mechanism of action is unknown. In this study, we examined toxicity of PRIMA-1 to three premalignant human colorectal adenoma cell lines (RG/C2, BR/C1, and AA/C1) and four colorectal carcinoma cell lines (DLD-1, SW480, LOVO, and HCT116) and its mechanism of action. It selectively induced apoptosis only in the mutant p53 premalignant and malignant colon cell lines, but was not toxic to the wild-type p53 premalignant and malignant colon cell lines. Using stable transfectants of temperature-sensitive p53 mutant Ala(143) in null p53 H1299 lung cancer cells, we found that PRIMA-1 induced significantly more apoptosis in cells with mutant p53 conformation (37 degrees C) than the wild-type p53 conformation (32.5 degrees C). Cell cycle analysis indicated that its inhibition of cell growth was correlated with induction of G(2) arrest. Western blot analysis showed PRIMA-1 increased p21 and GADD45 expression selectively in the mutant p53 cells. However, Fas, Bcl-2 family proteins, and caspases were not involved in PRIMA-1-induced cell death. The c-Jun-NH(2)-kinase (JNK) inhibitor SP 600125, but not p38 mitogen-activated protein kinase inhibitor SB 203580 or extracellular signal-regulated kinase inhibitor PD 98059, blocked PRIMA-1-induced apoptosis. Transfection with a dominant-negative phosphorylation mutant JNK, but not a dominant-negative p38 or wild-type JNK, inhibited PRIMA-1-induced cell death, suggesting that the JNK pathway plays an important role in PRIMA-1-induced apoptosis. PRIMA-1 is a highly selective small molecule toxic to p53 mutant cells and may serve as a prototype for the development of new p53-targeting agents for therapy of premalignant and malignant cells.

The role of alpha-helical structure in p53 peptides as a determinant for their mechanism of cell death: necrosis versus apoptosis.

Peptides derived from the N-terminal and C-terminal regions of the p53 tumor suppressor protein, linked to the membrane transduction domain of Antennapedia, have both been found to have significant cytotoxic effects selectively in human cancer cells. However, the N-terminal and C-terminal p53 peptides apparently display very different mechanisms for their anticancer effects. These differential effects can be attributed to dissimilar abilities to form distinctive 3-dimensional structures in extracellular-matrix-like aqueous solution that enable unique and selective cancer cell membrane penetration and effect. N-terminally based p53 peptides, with their ability to form distinctive S-shaped helix-loop-helix structures, are able to rapidly disrupt cancer cell membranes via toroidal-like pore formation causing necrosis; conversely, C-terminally based p53 peptides, due to their more random coil configuration, can be transduced across cancer cell membranes and bind to its intracellular target to cause a Fas pathway mechanism of apoptosis.

The use of fluorescent probes in the study of reactive oxygen species in pancreatic cancer cells.

The use of fluorescent probes can be an easy and quick method to analyze whether or not reactive oxygen species (ROS) are involved in a particular cellular process or the result of a particular drug treatment. ROS activate a variety of cell signaling and death pathways including apoptosis and necrosis (Raha and Robinson. Am J Med Gen 106:62-70, 2001). Here we describe a number of different probes, their specificities, advantages and limitations, as well as useful techniques important for their use.

Redirecting apoptosis to aponecrosis induces selective cytotoxicity to pancreatic cancer cells through increased ROS, decline in ATP levels, and VDAC.

Pancreatic cancer cell lines with mutated ras underwent an alternative form of cell death (aponecrosis) when treated concomitantly with clinically achievable concentrations of arsenic trioxide, ascorbic acid, and disulfiram (Antabuse; AAA). AAA's major effects are mediated through generation of intracellular reactive oxygen species (ROS) and more than 50% decline in intracellular ATP. N-acetyl cysteine and a superoxide dismutase mimetic prevented aponecrosis and restored intracellular ATP levels. DIDS (4,4'-diisothiocyanatostilbene-2, 2' disulfonic acid), the pan- Voltage-Dependent Anion Channel (VDAC), -1, 2, 3 inhibitor and short hairpin RNA (shRNA) to VDAC-1 blocked cell death and ROS accumulation. In vivo exposure of AAA led to a 62% reduction in mean tumor size and eliminated tumors in 30% of nude mice with PANC-1 xenografts. We concluded that early caspase-independent apoptosis was shifted to VDAC-mediated "targeted" aponecrosis by the addition of disulfiram to arsenic trioxide and ascorbic acid. Conceptually, this work represents a paradigm shift where switching from apoptosis to aponecrosis death pathways, also known as targeted aponecrosis, could be utilized to selectively kill pancreatic cancer cells resistant to apoptosis.

Capecitabine and temozolomide (CAPTEM) for metastatic, well-differentiated neuroendocrine cancers: The Pancreas Center at Columbia University experience.

PURPOSE: We evaluated the efficacy and safety of capecitabine and temozolomide (CAPTEM) in patients with metastatic neuroendocrine tumors (NETs) to the liver. This regimen was based on our studies with carcinoid cell lines that showed synergistic cytotoxicity with sequence-specific dosing of 5-fluorouracil preceding temozolomide (TMZ). METHODS: A retrospective review was conducted of 18 patients with NETs metastatic to the liver who had failed 60 mg/month of Sandostatin LAR™ (100%), chemotherapy (61%), and hepatic chemoembolization (50%). Patients received capecitabine at 600 mg/m(2) orally twice daily on days 1-14 (maximum 1,000 mg orally twice daily) and TMZ 150-200 mg/m(2) divided into two doses daily on days 10-14 of a 28-day cycle. Imaging was performed every 2 cycles, and serum tumor markers were measured every cycle. RESULTS: Using RECIST parameters, 1 patient (5.5%) with midgut carcinoid achieved a surgically proven complete pathological response (CR), 10 patients (55.5%) achieved a partial response (PR), and 4 patients (22.2%) had stable disease (SD). Total response rate was 61%, and clinical benefit (responders and SD) was 83.2%. Of four carcinoid cases treated with CAPTEM, there was 1 CR, 1 PR, 1 SD, and 1 progressive disease. Median progression-free survival was 14.0 months (11.3-18.0 months). Median overall survival from diagnosis of liver metastases was 83 months (28-140 months). The only grade 3 toxicity was thrombocytopenia (11%). There were no grade 4 toxicities, hospitalizations, opportunistic infections, febrile neutropenias, or deaths. CONCLUSIONS: CAPTEM is highly active, well tolerated and may prolong survival in patients with well-differentiated, metastatic NET who have progressed on previous therapies.

Selective depletion of mutant p53 by cancer chemopreventive isothiocyanates and their structure-activity relationships.

Isothiocyanates (ITCs) derived from cruciferous vegetables induce apoptosis in cancer cells. We demonstrate that certain naturally occurring ITCs selectively deplete mutant p53 but not the wild-type and do so via a transcription-independent mechanism. Direct p53 binding followed by conformational changes appears to be a mechanism by which mutant p53 is depleted. Structure-activity relationship studies (SARs) using naturally occurring and synthetic ITCs show that depletion is influenced by the ITC side-chain moiety. Furthermore, we show that cells with p53 mutations are more sensitive to cytotoxicity induced by phenethyl isothiocyanate (PEITC) than those with the wild-type protein. 2,2-Diphenylethyl ITC, a synthetic ITC, is one of the most potent depletors of mutant p53 studies and induces apoptosis to the greatest extent in mutant p53 breast cancer cells. Collectively, this study shows that mutant p53 depletion may be an important novel target for cancer chemoprevention and therapy by natural and synthetic ITCs.

Activation of targeted necrosis by a p53 peptide: a novel death pathway that circumvents apoptotic resistance.

Cancer cells escape apoptosis by intrinsic or acquired mechanisms of drug resistance. An alternative strategy to circumvent resistance to apoptosis could be through redirection into other death pathways, such as necrosis. However, necrosis is a nonspecific, nontargeted process resulting in cell lysis and inflammation of both cancer and normal cells and is therefore not a viable alternative. Here, we report that a C-terminal peptide of p53, called p53p-Ant, induced targeted necrosis only in multiple mutant p53 human prostate cancer lines and not normal cells, because the mechanism of cytotoxicity by p53p-Ant is dependent on the presence of high levels of mutant p53. Topotecan- and paclitaxel-resistant prostate cancer lines were as sensitive to p53p-Ant-induced targeted necrosis as parental lines. A massive loss of ATP pools and intracellular generation of reactive oxygen species was involved in the mechanism of targeted necrosis, which was inhibited by O(2)(.) scavengers. We hypothesize that targeted necrosis by p53p-Ant is dependent on mutant p53, is mediated by O(2)(.) loss and ATP, and can circumvent chemotherapy resistance to apoptosis. Targeted necrosis, as an alternative pathway for selective killing of cancer cells, may overcome the problems of nonspecificity in utilizing the necrotic pathway.

Selective induction of apoptosis through the FADD/caspase-8 pathway by a p53 c-terminal peptide in human pre-malignant and malignant cells.

A p53 C-terminal peptide (aa 361-382, p53p), fused at its C-terminus to the minimal carrier peptide of antennapedia (17 aa, Ant; p53p-Ant), induced rapid apoptosis in human cancer cells, via activation of the Fas pathway. We examined p53p-Ant mechanism of action, toxicity in various human normal, non-malignant, pre-malignant and malignant cancer cells and investigated its biophysical characteristics. p53p-Ant selectively induced cell death in only pre-malignant or malignant cells in a p53-dependent manner and was not toxic to normal and non-malignant cells. p53p-Ant was more toxic to the mutant p53 than wild-type p53 phenotype in H1299 lung cancer cells stably expressing human temperature-sensitive p53 mutant 143Ala. Surface plasmon resonance (BIACORE) analysis demonstrated that this peptide had higher binding affinity to mutant p53 as compared to wild-type p53. p53p-Ant induced-cell death had the classical morphological characteristics of apoptosis and had no features of necrosis. The mechanism of cell death by p53p-Ant was through the FADD/caspase-8-dependent pathway without the involvement of the TRAIL pathway, Bcl-2 family and cell cycle changes. Blocking Fas with antibody did not alter the peptide's effect, suggesting that Fas itself did not interact with the peptide. Transfection with a dominant-negative FADD with a deleted N-terminus inhibited p53p-Ant-induced apoptosis. Its mechanism of action is related to the FADD-induced pathway without restoration of other p53 functions. p53p-Ant is a novel anticancer agent with unique selectivity for human cancer cells and could be useful as a prototype for the development of new anti-cancer agents.