Heat Shock Proteins HSPA1 and HSP90AA1 Are Upregulated in Colorectal Polyps and Can Be Targeted in Cancer Cells by Anti-Inflammatory Oxicams with Arylpiperazine Pharmacophore and Benzoyl Moiety Substitutions at Thiazine Ring

Heat shock proteins HSPA1/Hsp70α and HSP90AA1/Hsp90α are crucial for cancer growth but their expression pattern in colorectal polyps or whether they can be modulated by oxicams is unknown. We quantified (RTqPCR) HSPA1 and HSP90AA1 expression in 50 polyp-normal pairs in relation to polyp malignancy potential and examined the effect of piroxicam, meloxicam and five novel analogues on HSPA1 and HSP90AA1 expression (mRNA/protein) in colorectal adenocarcinoma lines. HSPA1 and HSP90AA1 were upregulated in polyps by 3- and 2.9-fold. Expression ratios were higher in polyps with higher dysplasia grade and dominant villous growth pattern, mostly a result of diminished gene expression in normal tissue. Classic oxicams had negligible/non-significant effect on HSP expression. Their most effective analogue inhibited HSPA1 protein and gene by 2.5-fold and 5.7-fold in Caco-2 and by 11.5-fold and 6.8-fold in HCT116 and HSPA1 protein in HT-29 by 1.9-fold. It downregulated HSP90AA1 protein and gene by 1.9-fold and 3.7-fold in Caco-2 and by 2-fold and 5.0-fold in HCT116. HSPA1 and HSP90AA1 are upregulated in colorectal polyps reflecting their potential for malignancy. HSPA1 in cancer cells and, to lesser degree, HSP90AA1 can be reduced by oxicam analogues with thiazine ring substituted via propylene linker by arylpiperazine pharmacophore with fluorine substituents and by benzoyl moiety.


Introduction
Recent years have been associated with the rapid development of molecularly-targeted therapies. In colorectal cancer (CRC), they are principally addressed to patients with gross metastatic disease or primary tumors non-amenable for curative resection. For these patients, chemotherapy is a leading treatment option, although it is unsatisfactorily efficient, inducing resistance and characterized by high systemic toxicity. Potential molecular targets are researched from among pathways crucial for cancer growth, survival and progression [1]. Cancer-associated inflammation and its mediators fulfil these criteria as sustaining inflammation is included among key cancer characteristics, nurturing functionality of other hallmarks [2]. Consistently, a number of anti-inflammatory drugs, including oxicams-a class of nonsteroidal anti-inflammatory drugs (NSAIDs), has displayed chemopreventive
For RNA isolation and transcriptomic analysis, post-culture media were removed and cells were lyzed with TRIzol™ Reagent (Thermo Fisher Scientific). Collected lysates were stored at −80 • C.
For protein analysis, post-culture media were removed, cells were washed twice with phosphate-buffered saline (PBS) and scraped after addition of protease inhibitor cocktail (Complete Tablets EDTA-free; Roche Diagnostics, Mannheim, Germany) in PBS. Collected cell suspensions were kept frozen at −80 • C.

Transcriptomic Analysis
Total RNA was extracted from TRIzol™ cell lysates using phenol-chloroform extraction. Isolated RNA was purified using GenElute™ Mammalian Total RNA Miniprep Kit (Merck, Darmstadt, Germany) and genomic DNA contamination was avoided by oncolumn incubation with DNase I (Merck). cDNA was prepared following manufacturer's protocol from 1000 ng of RNA using iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA, USA). RNA concentration was determined using NanoDrop 2000 spectrophotometer (Thermo-Fisher Scientific).
For HSP analysis in patients' samples, previously obtained [34] and biobanked cDNA was used.
Data were tested for normality with Kolmogorov-Smirnov test and for variance homogeneity with Levene test. Expression data were log-transformed prior analysis. The t-test for paired observations was used to analyze patient-matched samples and oxicam treated and untreated cells. Unpaired analysis was conducted using t-test or one-way analysis of variance (ANOVA) with Students-Neuman-Keuls post hoc test. For data sets with non-homogeneous variances, the Kruskal-Wallis H test with Conover post hoc test was applied. Data on protein expression were compared using t-test for one mean.

HSPA1 and HSP90AA1 Expression in Patients with Colorectal Polyps
Pairwise analysis of patient-matched polyp and adjacent (normal) tissue showed a significant three-fold upregulation of both HSPA1 and HSP90AA1 expression in polyps (Table 3). To identify characteristics associated with the upregulation of HSPA1 and HSP90AA1 expression, the fold change (polyp-to-normal) as well as gene expression in polyps and polyp-adjacent macroscopically normal tissue was referred to polyp location, histological type, dysplasia grade, size, and number.
Fold change in HSP90AA1 (by 9.4-fold) but not HSPA1 expression was significantly greater in neoplasms located in the rectum as compared to right/left colon. It was associated with a tendency towards higher HSP90AA1 expression in rectal polyps accompanied by lower expression in rectal polyp-adjacent mucosa ( Figure 1). polyp-adjacent macroscopically normal tissue was referred to polyp location, histological type, dysplasia grade, size, and number. Fold change in HSP90AA1 (by 9.4-fold) but not HSPA1 expression was significantly greater in neoplasms located in the rectum as compared to right/left colon. It was associated with a tendency towards higher HSP90AA1 expression in rectal polyps accompanied by lower expression in rectal polyp-adjacent mucosa (Figure 1). Data presented on logarithmic scale as geometric means (red close squares) with 95% confidence interval (whiskers) and analyzed with t-test for independent samples. P/N, polyp-to-normal ratio; NRQ, normalized relative quantity.
Fold change in HSPA1 (ρ = 0.29, p = 0.042) and HSP90AA1 (ρ = 0.47, p < 0.001) expression depended on polyp type as it gradually increased along with increasing potential for malignancy. For HSPA1, there was no significant difference in mean fold change of expression between groups. HSPA1 was expressed in polyps at comparable level but its expression in adjacent tissue tended to decrease along with increasing potential for malignancy (ρ = −0.27, p = 0.059) (Figure 2).
Regrading HSP90AA1, polyps with the lowest potential for malignancy, hyperplastic polyps and tubular adenomas, had significantly lower polyp-to normal expression ratio than polyps with polyps with higher potential for malignancy-tubulo-villous adenomas and villous adenomas/adenocarcinomas (ρ = 0.47, p < 0.001). It was associated with changes in gene expression in both polyps and adjacent tissue. In polyps, HSP90AA1 expression increased along with potential for malignancy (ρ = 0.34, p = 0.015) while in adjacent tissue-it decreased (ρ = −0.30, p = 0.035) (Figure 2). Data presented on logarithmic scale as geometric means (red close squares) with 95% confidence interval (whiskers) and analyzed with t-test for independent samples. P/N, polyp-to-normal ratio; NRQ, normalized relative quantity.
Fold change in HSPA1 (ρ = 0.29, p = 0.042) and HSP90AA1 (ρ = 0.47, p < 0.001) expression depended on polyp type as it gradually increased along with increasing potential for malignancy. For HSPA1, there was no significant difference in mean fold change of expression between groups. HSPA1 was expressed in polyps at comparable level but its expression in adjacent tissue tended to decrease along with increasing potential for malignancy (ρ = −0.27, p = 0.059) (Figure 2).
Regrading HSP90AA1, polyps with the lowest potential for malignancy, hyperplastic polyps and tubular adenomas, had significantly lower polyp-to normal expression ratio than polyps with polyps with higher potential for malignancy-tubulo-villous adenomas and villous adenomas/adenocarcinomas (ρ = 0.47, p < 0.001). It was associated with changes in gene expression in both polyps and adjacent tissue. In polyps, HSP90AA1 expression increased along with potential for malignancy (ρ = 0.34, p = 0.015) while in adjacent tissue-it decreased (ρ = −0.30, p = 0.035) (Figure 2).
Fold change in HSPA1 expression between polyp and normal tissue was significantly greater in patients with high grade dysplasia or adenocarcinomas in polyps. It was associated with lower gene expression in their polyp-adjacent tissue while the expression in polyp did not differ depending on dysplasia grade ( Figure 3).  Fold change in HSPA1 expression between polyp and normal tissue was significantly greater in patients with high grade dysplasia or adenocarcinomas in polyps. It was associated with lower gene expression in their polyp-adjacent tissue while the expression in polyp did not differ depending on dysplasia grade ( Figure 3).
Likewise, fold change in HSP90AA1 expression tended to be greater in patients with high grade polyps or adenocarcinomas due to significantly lower expression in normal polyp-adjacent mucosa ( Figure 3). Likewise, fold change in HSP90AA1 expression tended to be greater in patients with high grade polyps or adenocarcinomas due to significantly lower expression in normal polyp-adjacent mucosa ( Figure 3).
Neither HSPA1 nor HSP90AA1 fold change differed significantly with respect to polyp size but HSPA1 expression decreased along with increasing size, significantly so, in both polyps (ρ = −0.39, p = 0.006) and adjacent tissue (ρ = −0.39, p = 0.05). Comparison of medians indicated that gene expression in the largest polyps was significantly lower as compared to the smallest in polyps and adjacent tissue and also as compared to medium size in case of polyps ( Figure 4). Fold change in HSPA1 expression between polyp and normal tissue was significantly greater in patients with high grade dysplasia or adenocarcinomas in polyps. It was associated with lower gene expression in their polyp-adjacent tissue while the expression in polyp did not differ depending on dysplasia grade ( Figure 3).
Likewise, fold change in HSP90AA1 expression tended to be greater in patients with high grade polyps or adenocarcinomas due to significantly lower expression in normal polyp-adjacent mucosa (Figure 3). Neither HSPA1 nor HSP90AA1 fold change differed significantly with respect to polyp size but HSPA1 expression decreased along with increasing size, significantly so, in both polyps (ρ = −0.39, p = 0.006) and adjacent tissue (ρ = −0.39, p = 0.05). Comparison of medians indicated that gene expression in the largest polyps was significantly lower as compared to the smallest in polyps and adjacent tissue and also as compared to medium size in case of polyps (Figure 4).  Neither HSPA1 nor HSP90AA1 fold change differed significantly with respect to polyp size but HSPA1 expression decreased along with increasing size, significantly so, in both polyps (ρ = −0.39, p = 0.006) and adjacent tissue (ρ = −0.39, p = 0.05). Comparison of medians indicated that gene expression in the largest polyps was significantly lower as compared to the smallest in polyps and adjacent tissue and also as compared to medium size in case of polyps ( Figure 4).  Data presented on logarithmic scale as geometric means (medians for HSPA1 in polyp and adjacent mucosa) (red close squares) with 95% confidence interval (whiskers) and analyzed with one-way ANOVA (Kruskal-Wallis H-test). Groups differing significantly (p < 0.05) in a post-hoc analysis (Conover test) were indicated by symbols of the same type (* orˆ). P/N, polyp-to-normal ratio; NRQ, normalized relative quantity.
Polyp-to-normal HSPA1 expression ratio tended to be higher in patients with single than multiple polyps (5.4 vs. 0.8, p = 0.093) but there was no significant difference in its expression in polyp (p = 0.203) or normal mucosa (p = 0.460).
Polyp-to-normal HSP90AA1 expression ratio did not differ significantly between patients with single than multiple polyps as well (4.5 vs. 1.2, p = 0.167) and there was no significant difference in its expression in polyp (p = 0.693) or normal mucosa (p = 0.101).

Effect of Oxicams on HSPA1 and HSP90AA1 Expression in Colorectal Adenocarcinoma Cell Lines Caco-2, HCT 116 and HT-29 3.2.1. HSPA1/Hsp70α and HSP90AA1/Hsp90α Proteins
To investigate the ability of classic (piroxicam and meloxicam) and novel analogues (compounds #1-5) to modulate HSPA1 and HSP90AA1 protein expression, Caco-2, HCT 116, and HT-29 were treated with 50 and 200 µM drug concentration for 48 h and cell protein content was analyzed by Western-blotting.
Regarding HSPA1 and classic drugs, there was large variation in cell response and none of the observed effects was statistically significant ( Figure 5).
Regarding HSPA1 and classic drugs, there was large variation in cell response and none of the observed effects was statistically significant ( Figure 5).
Polyp-to-normal HSPA1 expression ratio tended to be higher in patients with single than multiple polyps (5.4 vs. 0.8, p = 0.093) but there was no significant difference in its expression in polyp (p = 0.203) or normal mucosa (p = 0.460).
Polyp-to-normal HSP90AA1 expression ratio did not differ significantly between patients with single than multiple polyps as well (4.5 vs. 1.2, p = 0.167) and there was no significant difference in its expression in polyp (p = 0.693) or normal mucosa (p = 0.101).

Effect of Oxicams on HSPA1 and HSP90AA1 Expression in Colorectal Adenocarcinoma Cell Lines Caco-2, HCT 116 and HT-29
3.2.1. HSPA1/Hsp70α and HSP90AA1/Hsp90α Proteins To investigate the ability of classic (piroxicam and meloxicam) and novel analogues (compounds #1-5) to modulate HSPA1 and HSP90AA1 protein expression, Caco-2, HCT 116, and HT-29 were treated with 50 and 200 µM drug concentration for 48 h and cell protein content was analyzed by Western-blotting.
Regarding HSPA1 and classic drugs, there was large variation in cell response and none of the observed effects was statistically significant ( Figure 5).

HSPA1 and HSP90AA1 Transcripts
Transcriptional analysis was conducted on HCT 116 and Caco-2 cells as more responsive to oxicam treatment. To investigate the dose-dependent effect of oxicams on HSPA1 and HSP90AA1 gene expression, cells were treated with 5, 50 and 200 µM drug concentration for 24 h and gene expression was analyzed with RTqPCR. In addition, the effect of time was determined in 6 and 24-h cultures treated with 200 µM oxicams and in 24 and 72-h cultures treated with 5 µM oxicams.
Classic oxicams had negligible/non-significant effect on HSPA1 (Figure 9) and HSP90AA1 expression ( Figure 10). tration for 24 h and gene expression was analyzed with RTqPCR. In addition, the effect of time was determined in 6 and 24-h cultures treated with 200 µM oxicams and in 24 and 72-h cultures treated with 5 µM oxicams.

Discussion
Discerning the molecular mechanisms underlying drugs' anti-tumor activity and detailed understanding of expression patterns and relevance of prospective targets is prerequisite for developing safe and effective molecular therapies. It is also crucial in enabling design of chemicals with improved characteristics [36]. Owing to their central role in proteostasis and cell signaling, heat shock proteins constitute a unique target for antineoplastic therapies holding promise to circumvent cancer plasticity [28,29]. Here, we showed that both HSPA1 and HSP90AA1 transcripts are upregulated, to the same degree, in colorectal polyps as compared to polyp-adjacent tissue.
HSPA1/Hsp70 is a prototypical and an inducible member of HSP70 family, which guards cancer cells against stress-induced proteotoxicity. Consistently, it is overexpressed in number of cancers, although clinical data regarding CRC are, as observed by Gao at al. [37], surprisingly scanty. Lazaris et al. [38] showed 77% of colorectal tumor samples to

Discussion
Discerning the molecular mechanisms underlying drugs' anti-tumor activity and detailed understanding of expression patterns and relevance of prospective targets is prerequisite for developing safe and effective molecular therapies. It is also crucial in enabling design of chemicals with improved characteristics [36]. Owing to their central role in proteostasis and cell signaling, heat shock proteins constitute a unique target for antineoplastic therapies holding promise to circumvent cancer plasticity [28,29]. Here, we showed that both HSPA1 and HSP90AA1 transcripts are upregulated, to the same degree, in colorectal polyps as compared to polyp-adjacent tissue. HSPA1/Hsp70 is a prototypical and an inducible member of HSP70 family, which guards cancer cells against stress-induced proteotoxicity. Consistently, it is overexpressed in number of cancers, although clinical data regarding CRC are, as observed by Gao at al. [37], surprisingly scanty. Lazaris et al. [38] showed 77% of colorectal tumor samples to contain at least 10% of neoplastic cells with Hsp70-immunoreactivity. The immunoreactivity was positively correlated with cancer aggressiveness, as it increased along with tumor dedifferentiation, and inversely with patients' survival. The association between Hsp70 protein expression and patients' prognosis, but not tumor grade or cancer stage, has subsequently been confirmed by others [39][40][41]. Likewise, Hsp70 elevation in serum has been linked with poor prognosis and the disease advancement [24]. DLD-1 cells with downregulated HSPA1 expression responded to a treatment with a heat shock responseinducing agent with a 3-fold upregulation [42]-which corresponds with a degree of average elevation in gene expression in polyps observed in clinical samples evaluated in current study. Mechanistically, HSPA1/Hsp70 expression improved migratory properties of DLD-1 cells and its knockdown reduced by several-fold the expression of Snail, Snug and Twist, the E-cadherin suppressors and inductors of epithelial-mesenchymal transition [42]. Others have shown selective HSPA1 knockout to enhance cancer cell immunogenicity [43] and induce cell death in xenografts independent from caspase/Bcl-2 pathway [44].
To the best of our knowledge, HSPA1 expression in precancerous colorectal lesions has not been addressed. Herein, HSPA1 expression rate between polyp and adjacent tissue increased significantly along with increasing potential for malignancy. Interestingly, however, the rising trend resulted from diminishing expression in histologically normal polyp-adjacent tissue while polyp expression remined comparable in hyperplastic/tubular polyps with low potential for malignancy, tubulo-villous with medium potential for malignancy and villous polyps/adenocarcinomas in polyps with the highest potential for malignancy. Likewise, HSPA1 expression rate was significantly higher in polyps with high grade dysplasia, again owing to a drop in the gene expression in adjacent tissue. Of note, alterations in molecular landscape of histologically and morphologically normal tissue surrounding colorectal adenocarcinomas have repeatedly been demonstrated [19,34,45] and suspected of contributing to synchronous tumors and/or cancer recurrence after curative resection [46]. Apart from polyp type and dysplasia grade, malignant potential is believed to correlate directly with polyp size [47]. The rate of HSPA1 expression was not associated with polyp size in the examined cohort. However, unexpectedly, our results showed that gene expression decreased alongside increasing polyp size, both in polyp and polypadjacent tissue, which might imply a protective role for HSPA1/Hsp70 prior neoplastic transformation. Low level of gene expression may increase susceptibility of cellular protein to stress-induced damage and thus facilitate transformation. However, contradicting the notion, transforming potential of HSPA1/Hsp70 overexpression, and not downregulation, has been demonstrated in Apc Min/+ mouse model of CRC. The model is characterized by development of adenomas in the small and large intestine. Tao et al. [41] showed that the loss of Hsp70 reduces the number and size of adenomas and decreases their proliferation rate and resistance to apoptosis as compared to Hsp70-expressing mice. Mechanistically, loss of Hsp70 attributed to enhanced degradation of β-catenin without an effect on its gene expression while Hsp70 expression activated of Akt, ERK, and p38/MAPK pathways [41]. Likewise, HSPA1/Hsp70 has been necessary for neoplastic transformation of mammary epithelial cells induced by Her2 oncogene [48]. Taken together, in vitro and animal-based findings evoked an interest in HSPA1/Hsp70 as potential target for chemoprevention in addition to anti-tumor therapies.
Therefore, we evaluated the ability of classic, piroxicam and meloxicam, as well as novel oxicam analogues to modulate HSPA1/Hsp70 expression in HCT 116, Caco-2 and HT-29 cells. Regarding classic oxicams, neither protein nor mRNA expression were significantly affected as there was rather high variability in cell response between biological replicates. Unlike classic oxicams, their novel analogues were effective in downregulating HSPA1 already at 50 µM concentration at protein and mRNA level. HCT 116 cells were the most sensitive to oxicam analogues and the line has been claimed to not express COX2 [49]. Therefore, it is likely that HSP-inhibiting effect is not mediated by drug ability to inhibit enzyme activity.
Structurally, all tested analogues differ from classic drugs with arylpiperazine pharmacophore and benzoyl moiety substitutions at thiazine ring. It has previously been noted that such modification enhances anti-inflammatory properties of the drug, owing to high electron-withdrawing properties of arylpiperizine pharmacophore [50]. Indeed, the presence of this moiety has allowed novel analogues to regulate expression of enzymes involved in L-arginine/nitric oxide pathway [19] as well as monocyte/macrophageassociated chemokines (manuscript submitted). Like in case of L-arginine/nitric oxide pathway enzymes and chemokines, compounds with 3-carbon propylene linker between nitrogen atoms of thiazine and piperazine rings (compounds #1, #2 and #3) were generally more effective in downregulating HSPA1 than those with 2-carbon oxyethylene linker (compounds #4 and #5). Its presence was crucial for gene downregulation in Caco-2 cells. Of the evaluated analogues, compounds #2 and #3 seem to be the most effective, consistently downregulating both HSPA1 protein and mRNA in both cell lines, with compound #3 significantly downregulating HSPA1 also in HT-29 cells. They are distinguished from compound #1 by the presence of fluoro-substituents at arylpiperazine ring, likely to further enhance the electron-withdrawing and thus anti-inflammatory properties of the pharmacophore. Of note, as HSPs may engage receptors involved in NFκB activation [27], some anti-inflammatory effects exerted by novel oxicams containing arylpiperazine pharmacophore, but not classic drugs from this group, e.g., chemokine downregulation, might be mediated by their inhibitory effect on HSPA1. Intriguingly, a biphasic type of response was observed in case of HSPA1 mRNA expression in Caco-2 cells, previously noted also in case of L-arginine/nitric oxide pathway enzymes [19] and chemokines (manuscript submitted). The gene downregulation after 24-h incubation with lower analogue concentration (5 and/or 50 µM) or 6-h incubation with 200 µM but its upregulation following 24-h incubation with 200 µM drugs.
Tested oxicam analogues, but not classic drugs, were effective also in downregulating expression of HSP90AA1. Like for HSPA1, HCT 116 cells were more responsive and compounds #2 and #3 more efficient than other ones, but the effect seemed to be generally slightly less marked. The inhibition was less evident at protein level. HSP90AA1 protein was significantly decreased upon treatment of Caco-2 with compounds #3-5 and HCT 116 with compounds #2 and #3. Regarding clinical samples, HSP90AA1 was upregulated in polyps to the very same degree as HSPA1. Like HSPA1, its expression ratio (polypto-normal) was dependent on polyp malignancy potential. It was higher in polyps with dominant villous growth pattern and high grade of dysplasia. In case of dysplasia grade, the expression pattern resembled that of HSPA1: the higher expression ratio resulted from lower gene expression in adjacent tissue while polyp expression did not differ between polyps with low and high dysplasia grade. In case of polyp type, the HSP90AA1 expression rate increasing along with growing contribution of villous growth pattern resulted not from both decreasing gene expression in adjacent tissue and from increasing expression in polyp. Unlike HSPA1, HSP90AA1 expression was not associated with polyp size but was higher in case of rectal than colonic polyps.
Neoplastic transformation is believed to occur as a result of overwhelming protective Hsp90 capacity during periods of high cellular stress [23]. In this respect, diminishing HSP90AA1 expression in normal polyp-adjacent tissue along with increasing polyp potential for malignancy renders cellular proteins susceptible to stressors, which may translate into creating tumor-promoting environment. In transformed cells, Hsp90 acts to preserve malignant phenotype by facilitating accumulation of beneficial while suppressing mutations lethal for cancer cells [23]. Therefore, higher HSP90AA1 expression in polyps with greater potential for malignancy, observed in evaluated clinical samples, agrees well with Hsp90 function as a key facilitator of unrestrained growth owned to Hsp90-mediated stabilization of proteins involved in proliferation [23]. In the light of significance of this heat shock protein for cancer adaptability to endogenous (e.g., oxidative and metabolic stress, hypoxia) and exogeneous (e.g., chemo/radiotherapy) stressors as well as its role in stabilization of oncogenic proteins, particularly those facilitating cancer growth, invasion and metastasis [23,51], markedly weaker effect of oxicam analogues on HSP90AA1 protein is disappointing. Targeting Hsp90 in CRC is of particular interest due to high incidence of KRAS mutations and constitutive activation of Ras/Raf/MEK/Erk signaling, pathways susceptible to Hsp90 inhibition. Moreover, Hsp90 inhibition sensitizes colorectal cancer cells to oxaliplatin and the underlying molecular mechanisms involves hampering NFκB signaling [52]. The ability of investigated oxicams to downregulate gene but not protein expression might imply an involvement of counteractive posttranslational mechanisms, which warrants further investigation.

Conclusions
The expression of HSPA1 and HSP90AA1, key heat shock proteins involved in facilitating neoplastic transformation and cancer development, is altered already in precancerous colorectal lesions and surrounding tissue, to degree dependent on polyp potential for malignancy. In colorectal cancer cells, the number of HSPA1 and HSP90AA1 transcripts as well as the amount of HSPA1 and, to lesser degree, HSP90AA1 protein can be altered by novel oxicam analogues containing arylpiperazine pharmacophore and benzoyl moiety substitutions at thiazine ring instead of methyl substituent at position 2 and 2-peridocarbamoyl substituent at position 3, respectively. Analogue efficacy was dependent on the presence of 3-carbon propylene linker between thiazine and piperazine nitrogens and on fluorine substituents at arylpiperazine pharmacophore.